Method and kit for simultaneous detection of target nucleic acids and target proteins

Abstract

A method for simultaneously detecting a target nucleic acid and a target protein in a biological sample, comprising treating the biological sample with a cross-linking agent after incubating the biological sample with a primary antibody that detects the target protein and before detecting the target nucleic acid by in situ hybridization.

Description

Detailed description of the invention

[0001] [Technical Field] 1. Cross-reference of related applications This application claims priority and interest in U.S. Provisional Patent Application No. 63 / 021,632, filed on 7 May 2020, which is incorporated herein by reference in its entirety for all purposes.

[0002] 2. Field Embodiments of this disclosure include methods for preparing biological samples for the simultaneous detection of target nucleic acids and target proteins. Methods for the simultaneous detection of target nucleic acids and target proteins, as well as kits for carrying out such methods, are also provided.

[0003] [Background technology] 3.Background technology In situ hybridization (ISH) is a widely used molecular biological technique to detect and locate specific sequences within cell or tissue sections while preserving the cell and tissue background. Therefore, ISH enables spatial and temporal visualization and quantification of gene expression within cells and tissues, which has useful applications in research and diagnostics. See Hu et al., Biomarker Research 2(1):1-13(2014), Ratan et al., Cureus 9(6):e1325(2017), and Weier et al., Expert Review of Molecular Diagnostics 2(2):109-119(2002).

[0004] Immunohistochemistry (IHC) and immunocytochemistry (ICC) are also powerful techniques used to detect and locate specific proteins within tissue sections and cells while maintaining spatial resolution and cytological background. Like ISH, IHC and ICC have broad and complementary applications in research and diagnostics. (See Shi et al., Journal of Histochemistry & Cytochemistry 59(11):13-32(2011)). For example, both techniques provide researchers with insights into the identity and state of cells.

[0005] A multi-omics strategy is necessary for the complete characterization of complex intercellular interactions within tissues or cells. For example, the detection and analysis of transcriptomics and proteomics information is useful when investigating composite tissues, revealing cell type-specific gene expression (see Vanlandewijck et al., Nature 554(7693):475-482 (2018), Stempl et al., the Journal of Molecular Diagnostics 14(1):22-29 (2014)), identifying the cellular origin of secreted proteins (see Liou et al., Cell Reports 19(7):1322-1333 (2017)), and visualizing the spatial configuration of various cell types and their interactions. To fully and accurately characterize cells and tissues, nucleic acids and proteins must be detected simultaneously in the same tissue sample in a spatially distinguishable manner.

[0006] Despite the crucial role of dual detection, it presents challenges in obtaining high-quality signals for nucleic acids and proteins in the same sample. Therefore, there is a significant demand for novel methods capable of detecting both signals, such as simultaneously performing ISH and IHC / ICC in a single sample without interference. This disclosure addresses these and other needs.

[0007] 4. Outline of the Invention In one embodiment, the Specified provides a method for preparing a biological sample for the simultaneous detection of a target nucleic acid and a target protein, comprising: (i) incubating the biological sample with a primary antibody; (ii) treating the biological sample with a crosslinking agent after (i); and (iii) detecting the target nucleic acid by in situ hybridization after (ii).

[0008] In some embodiments, the method further includes treating the biological sample with a crosslinking agent and then with a protease before detecting the target nucleic acid by in situ hybridization. In other embodiments, the method further includes incubating the biological sample with a secondary antibody or other labeling method after detecting the target nucleic acid by in situ hybridization. In some embodiments, the target nucleic acid is RNA or DNA.

[0009] In some embodiments, the biological sample is a tissue sample or derived from a tissue sample. In other embodiments, the biological sample is a blood sample or derived from a blood sample. In yet another embodiment, the biological sample is a cytological sample or derived from a cytological sample. In yet another embodiment, the biological sample is a cultured cell or exosome-containing sample.

[0010] In some embodiments, the crosslinking agent in step (ii) is a fixative. In certain embodiments, the fixative is neutral buffered formalin (NBF). In one embodiment, the neutral buffered formalin is 10% neutral buffered formalin.

[0011] In some embodiments, the process of treating the biological sample with a crosslinking agent lasts for approximately 15 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, the process of treating the biological sample with a crosslinking agent is carried out at approximately 4°C, room temperature, approximately 40°C, or approximately 60°C.

[0012] In some embodiments, the process of treating the biological sample with a crosslinking agent is carried out at approximately 4°C for approximately 2 hours. In other embodiments, the process of treating the biological sample with a crosslinking agent is carried out at 4°C for approximately 16 to 18 hours.

[0013] In some embodiments, the process of treating the biological sample with a crosslinking agent is carried out at room temperature for 15 minutes. In other embodiments, the process of treating the biological sample with a crosslinking agent is carried out at room temperature for approximately 30 minutes. In yet another embodiment, the process of treating the biological sample with a crosslinking agent is carried out at room temperature for approximately 60 minutes.

[0014] In some embodiments, the process of treating the biological sample with a crosslinking agent is carried out at approximately 40°C for approximately 15 minutes. In other embodiments, the process of treating the biological sample with a crosslinking agent is carried out at approximately 40°C for approximately 30 minutes. In yet another embodiment, the process of treating the biological sample with a crosslinking agent is carried out at approximately 40°C for approximately 60 minutes.

[0015] In some embodiments, the process of treating the biological sample with a crosslinking agent is carried out at approximately 60°C for approximately 15 minutes. In other embodiments, the process of treating the biological sample with a crosslinking agent is carried out at approximately 60°C for approximately 30 minutes. In yet another embodiment, the process of treating the biological sample with a crosslinking agent is carried out at approximately 60°C for approximately 60 minutes.

[0016] In another aspect, provided herein is a method for simultaneously detecting a target nucleic acid and a target protein in a biological sample, comprising: (i) incubating the biological sample with a primary antibody; (ii) treating the biological sample with a crosslinking agent; (iii) treating the biological sample with a protease; (iv) detecting the target nucleic acid by in situ hybridization; and (v) detecting the target protein by incubating the biological sample with a secondary antibody or other labeling method.

[0017] In some embodiments, the target nucleic acid is RNA or DNA.

[0018] In some embodiments, the step of detecting the target nucleic acid by in situ hybridization comprises: (i) providing one or more target probes capable of hybridizing to the target nucleic acid; (ii) providing a signal generating complex capable of hybridizing to the one or more target probes, wherein in certain embodiments, the signal generating complex comprises a nucleic acid component capable of hybridizing to the one or more target probes and a labeled probe; (iii) hybridizing the target nucleic acid to the one or more target probes; and (iv) capturing the signal generating complex to the one or more target probes, thereby capturing the signal generating complex to the target nucleic acid. In certain embodiments, each of the one or more target probes comprises a target (T) section and a label (L) section. In certain embodiments, the T section is a nucleic acid sequence complementary to a section on the target nucleic acid, and the L section is a nucleic acid sequence complementary to a section on the nucleic acid component of the signal generating complex. In certain embodiments, the T section of the one or more target probes is complementary to a non-overlapping region of the target nucleic acid, and the L section of the one or more target probes is complementary to a non-overlapping region of the nucleic acid component of the signal generating complex.

[0019] In some embodiments, the method further includes providing an immunohistochemical label that can be bound to a secondary antibody for detecting a target protein. In other embodiments, the secondary antibody is pre-labeled.

[0020] In some embodiments, the biological sample is a tissue sample or derived from a tissue sample. In other embodiments, the biological sample is a blood sample or derived from a blood sample. In yet another embodiment, the biological sample is a cytological sample or derived from a cytological sample. In yet another embodiment, the biological sample is a cultured cell or exosome-containing sample.

[0021] In some embodiments, the crosslinking agent is a fixative. In certain embodiments, the fixative is neutral buffered formalin. In one embodiment, the neutral buffered formalin is 10% neutral buffered formalin.

[0022] In some embodiments, the process of treating the biological sample with a crosslinking agent lasts for approximately 15 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, the process of treating the biological sample with a crosslinking agent is carried out at approximately 4°C, room temperature, approximately 40°C, or approximately 60°C.

[0023] In some embodiments, the process of treating the biological sample with a crosslinking agent is carried out at approximately 4°C for approximately 2 hours. In other embodiments, the process of treating the biological sample with a crosslinking agent is carried out at approximately 4°C for approximately 16 to 18 hours.

[0024] In some embodiments, the process of treating the biological sample with a crosslinking agent is carried out at room temperature for about 15 minutes. In other embodiments, the process of treating the biological sample with a crosslinking agent is carried out at room temperature for about 30 minutes. In yet another embodiment, the process of treating the biological sample with a crosslinking agent is carried out at room temperature for about 60 minutes.

[0025] In some embodiments, the process of treating the biological sample with a crosslinking agent is carried out at approximately 40°C for approximately 15 minutes. In other embodiments, the process of treating the biological sample with a crosslinking agent is carried out at approximately 40°C for approximately 30 minutes. In yet another embodiment, the process of treating the biological sample with a crosslinking agent is carried out at approximately 40°C for approximately 60 minutes.

[0026] In some embodiments, the process of treating the biological sample with a crosslinking agent is carried out at approximately 60°C for approximately 15 minutes. In other embodiments, the process of treating the biological sample with a crosslinking agent is carried out at approximately 60°C for approximately 30 minutes. In yet another embodiment, the process of treating the biological sample with a crosslinking agent is carried out at approximately 60°C for approximately 60 minutes.

[0027] In some embodiments, the method is used to map the spatial configuration in composite tissues. In certain embodiments, the composite tissue is tumor tissue. In other embodiments, the method is used to detect denatured gene expression in biological samples from diseased models. In yet another embodiment, the method is used to validate novel antibodies.

[0028] In another embodiment, this specification provides a kit for the simultaneous detection of a target nucleic acid and a target protein in a biological sample, comprising (i) a crosslinking agent and (ii) instructions for use indicating that the crosslinking agent is used after the biological sample has been incubated with a primary antibody for detecting the target protein. In some embodiments, the kit further comprises a protease. In some embodiments, the kit further comprises a drug for detecting the target nucleic acid and / or a drug for detecting the target protein.

[0029] In another embodiment, this specification provides a kit for simultaneously detecting a target nucleic acid and a target protein in a biological sample, comprising (i) a crosslinking agent and (ii) a protease. In some embodiments, the kit further includes instructions indicating that the crosslinking agent is used before the protease and that the crosslinking agent is used after a primary antibody for detecting the target protein. In some embodiments, the kit further includes an agent for detecting the target nucleic acid and / or an agent for detecting the target protein. In some embodiments, the target nucleic acid is RNA or DNA.

[0030] In some embodiments, the instructions further indicate that the biological sample is treated with the crosslinking agent for approximately 15 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, the instructions further indicate that the biological sample is treated with the crosslinking agent at approximately 4°C, room temperature, 40°C, or 60°C.

[0031] In some embodiments, the instructions further indicate that the biological sample is treated with the crosslinking agent for about 2 hours at about 4°C. In other embodiments, the instructions further indicate that the biological sample is treated with the crosslinking agent for about 16 to 18 hours at about 4°C.

[0032] In some embodiments, the instructions further indicate that the biological sample is treated with the crosslinking agent for about 15 minutes at room temperature. In other embodiments, the instructions further indicate that the biological sample is treated with the crosslinking agent for about 30 minutes at room temperature. In yet another embodiment, the instructions further indicate that the biological sample is treated with the crosslinking agent for about 60 minutes at room temperature.

[0033] In some embodiments, the instructions further indicate that the biological sample is treated with the crosslinking agent at approximately 40°C for approximately 15 minutes. In other embodiments, the instructions further indicate that the biological sample is treated with the crosslinking agent at approximately 40°C for approximately 30 minutes. In yet another embodiment, the instructions further indicate that the biological sample is treated with the crosslinking agent at approximately 40°C for approximately 60 minutes.

[0034] In some embodiments, the instructions further indicate that the biological sample is treated with the crosslinking agent at approximately 60°C for approximately 15 minutes. In other embodiments, the instructions further indicate that the biological sample is treated with the crosslinking agent at approximately 60°C for approximately 30 minutes. In yet another embodiment, the instructions further indicate that the biological sample is treated with the crosslinking agent at approximately 60°C for approximately 60 minutes.

[0035] In some embodiments, a drug for detecting a target nucleic acid comprises one or more target probes that can hybridize to the target nucleic acid, and a signal-generating complex that can hybridize to one or more target probes, wherein in certain embodiments, the signal-generating complex comprises a nucleic acid component that can hybridize to one or more target probes, and a label probe. In certain embodiments, each of the one or more target probes comprises a target (T) section and a label (L) section. In certain embodiments, the T section is a nucleic acid sequence complementary to the section on the target nucleic acid, and the L section is a nucleic acid sequence complementary to the section on the nucleic acid component of the signal-generating complex. In certain embodiments, the T section of one or more target probes is complementary to a non-overlapping region of the target nucleic acid, and the L section of one or more target probes is complementary to a non-overlapping region of the nucleic acid component of the signal-generating complex.

[0036] In some embodiments, the kit further includes tools for obtaining biological samples. In certain embodiments, the biological sample is or is derived from a tissue sample. In certain embodiments, the biological sample is or is derived from a blood sample. In certain embodiments, the biological sample is or is derived from a cytological sample. In certain embodiments, the biological sample is a cultured cell or exosome-containing sample.

[0037] In some embodiments, the kit is used to map the spatial configuration in composite tissues. In certain embodiments, the composite tissue is tumor tissue. In other embodiments, the kit is used to detect denatured gene expression in biological samples from diseased models. In yet another embodiment, the kit is used to validate novel antibodies.

[0038] In another embodiment, a biological sample prepared by the above method is provided herein.

[0039] 5. Brief description of the drawing [Figure 1A] This shows a schematic diagram of the sequential ISH / IHC and integrated ISH / IHC codetection workflows. This diagram shows the steps of the sequential ISH-IHC workflow.

[0040] [Figure 1B] This shows a schematic diagram of the sequential ISH / IHC and integrated ISH / IHC codetection workflows. This diagram shows the steps of the integrated ISH / IHC codetection workflow.

[0041] [Figure 2] This shows that the IHC signal of CD20 was protected by crosslinking from stress caused by formamide-based reagents in hybridization buffer. A shows formalin-fixed paraffin-embedded (FFPE) human tonsil tissue sections stained with IHC using the Leica Bond Polymer Refine Detection Kit. B shows a section in which the IHC signal was reduced after exposure to hybridization buffer at room temperature for 30 minutes. C shows that the IHC signal was protected by crosslinking after primary antibody despite exposure to hybridization buffer.

[0042] [Figure 3] This shows that the resistance of the CD20 IHC signal to protease treatment was protected by crosslinking. A shows a human tonsil tissue section in which the CD20 protein was detected by IHC staining using the Leica Bond Polymer Refine Detection Kit. B shows the IHC signal reduced after exposure to the protease enzyme at 40°C for 15 minutes. C shows the IHC signal protected by crosslinking after primary antibody despite exposure to the protease enzyme.

[0043] [Figure 4] The IHC signal of CD8 was enhanced by crosslinking before hybridization buffer or protease treatment. A shows a human tonsil tissue section stained with IHC using the Leica Bond Polymer Refine Detection Kit to detect CD8. B shows the IHC signal that decreased after exposure to hybridization buffer at room temperature for 30 minutes. C shows the IHC signal that was enhanced by crosslinking after primary antibody despite exposure to hybridization buffer. D shows a human tonsil section stained with IHC using the Leica Bond Polymer Refine Detection Kit to detect CD8. E shows the IHC signal that decreased after exposure to protease enzyme at 40°C for 15 minutes. F shows the IHC signal that was enhanced by crosslinking after primary antibody despite exposure to protease enzyme.

[0044] [Figure 5] This shows that the ISH / IHC integrated co-detection workflow enables co-detection of the CD8 protein using human peptidyl prolyl isomerase B (Hs-PPIB) ISH staining. A shows a section of a human head and neck cancer sample from FFPE that has been IHC stained for CD8 using the Leica Bond Polymer Refine Detection kit. B shows ISH staining of Hs-PPIB using the RNAscope® 2.5 LS Red kit. C shows the ISH and IHC signals after the conventional procedure in which ISH and IHC are performed sequentially. D shows the enhanced ISH and IHC signals after the ISH / IHC integrated co-detection workflow.

[0045] [Figure 6] This shows that crosslinking is key to improving the IHC signal in the ISH / IHC integrated co-detection workflow. A shows a section of FFPE human tonsil tissue stained with IHC for CD20 using the Leica Bond Polymer Refine Detection kit, followed by the Green chromogen. B shows a section of FFPE human tonsil tissue stained with ISH for Hs-PPIB using the RNAscope® 2.5 LS Red kit. C shows a section of FFPE human tonsil tissue that underwent RNAscope® pretreatment, including 15 minutes of incubation with protease at 40°C before IHC staining, resulting in a reduction of the IHC signal. D shows a section of FFPE human tonsil tissue that underwent the ISH / IHC integrated co-detection workflow without the crosslinking step. E shows a section of FFPE human tonsil tissue that underwent the ISH / IHC integrated co-detection workflow with the crosslinking step.

[0046] [Figure 7] This shows that crosslinking at room temperature was effective when performed for 15 to 60 minutes. A shows sections of FFPE human gastric cancer tissue that were stained with ISH for Hs-PPIB detection and immediately followed by CD8 IHC detection using RNAscope® 2.5 LS Red and Leica Bond Polymer Refine Detection kits, respectively. B shows sections of FFPE human gastric cancer tissue that were crosslinked at room temperature for 15 minutes using the ISH / IHC integrated co-detection workflow. C shows sections of FFPE human gastric cancer tissue that were crosslinked at room temperature for 30 minutes using the ISH / IHC integrated co-detection workflow. D shows sections of FFPE human gastric cancer tissue that were crosslinked at room temperature for 60 minutes using the ISH / IHC integrated co-detection workflow.

[0047] [Figure 8] This shows that the IHC signal was preserved by crosslinking at the heated temperature. A shows a section of FFPE human gastric cancer tissue that was stained with ISH for Hs-PPIB detection and then immediately subjected to CD8 IHC detection. B shows a section of FFPE human gastric cancer tissue that was crosslinked at 40°C for 15 minutes using the ISH / IHC integrated co-detection workflow. C shows a section of FFPE human gastric cancer tissue that was crosslinked at 40°C for 30 minutes using the ISH / IHC integrated co-detection workflow. D shows a section of FFPE human gastric cancer tissue that was crosslinked at 40°C for 60 minutes using the ISH / IHC integrated co-detection workflow. E shows a section of FFPE human gastric cancer tissue that was crosslinked at 60°C for 15 minutes using the ISH / IHC integrated co-detection workflow. F shows a section of FFPE human gastric cancer tissue that was crosslinked at 60°C for 30 minutes using the ISH / IHC integrated co-detection workflow. Figure G shows sections of FFPE human gastric cancer tissue crosslinked at 60°C for 60 minutes using an ISH / IHC integrated co-detection workflow.

[0048] [Figure 9] This shows that the IHC signal was preserved by crosslinking at 4°C. A shows a section of FFPE human gastric cancer tissue that was stained with ISH for Hs-PPIB detection and then immediately subjected to CD8 IHC detection. B shows a section of FFPE human gastric cancer tissue that was crosslinked at room temperature for 30 minutes using the ISH / IHC integrated co-detection workflow. C shows a section of FFPE human gastric cancer tissue that was crosslinked at 4°C for 2 hours using the ISH / IHC integrated co-detection workflow. D shows a section of FFPE human gastric cancer tissue that was crosslinked overnight at 4°C using the ISH / IHC integrated co-detection workflow.

[0049] [Figure 10A] shows a typical workflow diagram of an image processing method for reducing background signal according to one embodiment of the present disclosure, and describes the image being processed.

[0050] [Figure 10B] shows a typical workflow diagram of an image processing method for reducing background signal according to one embodiment of the present disclosure, and describes the general steps of this method.

[0051] 6. Modes for Carrying Out the Invention This disclosure is partly based on the remarkable discovery that ISH and IHC can be effectively performed simultaneously with minimal interference by rearranging the experimental procedure and adding a step to crosslink proteins in the sample. In some embodiments, the incubation of the primary antibody and the secondary antibody are separated in order to preserve and improve the IHC and ISH signals. Furthermore, a crosslinking step follows incubation with the primary antibody (see Figure 1B), thereby protecting the binding of the primary antibody to the antigen from degradation (e.g., by protease treatment) or other interference from hybridization buffers and high-temperature incubation commonly used in ISH (see Figures 2A-2C, 3A-3C, and 4A-4F). This technique leads to enhanced simultaneous detection of nucleic acids and proteins.

[0052] 6.1 Definition As used herein, the terms “in situ hybridization” or “ISH” mean a technique for locating and visualizing a specific target nucleic acid, with preservation of the morphology of the source sample.

[0053] As used herein, the term “immunohistochemistry” or “IHC” generally refers to techniques for detecting proteins of a target using antibodies in a source sample (e.g., a tissue sample), with preservation of the morphology of the source sample. As used herein, the term “immunocytochemistry” or “ICC” generally refers to techniques for detecting proteins of a target using antibodies in a source sample (e.g., isolated or cultured cells, such as tissue culture cell lines in adherent or suspension), with preservation of the morphology of the source sample. Immunofluorescence (IF) means fluorescent labeling and is therefore also encompassed in the terms IHC and ICC. ICC, IHC, and IF assays can be used in conjunction with imaging methods of the present disclosure to facilitate quantitative and / or qualitative assessment of targets of a target in a sample, as further described herein. ICC, IHC, and IF assays can also be performed in conjunction with in situ hybridization as part of an integrated co-detection step for detecting targets of a target, which may further include the execution of imaging methods of the present disclosure.

[0054] As used herein, the term “simultaneously” or “concurrent” means the process of obtaining signals from different assays performed on the same biological sample, rather than on different samples. Therefore, it is not necessarily limited to the exact timing of data acquisition or signal detection from different assays. A reading from one assay may follow a reading from another.

[0055] As used herein, the term “crosslinking” means the process of joining two or more molecules together. “Crosslinking agent” or equivalent means a substance containing two or more chemically reactive ends that bond themselves to functional groups found in proteins and other molecules. Specifically, when the crosslinking agent is formaldehyde or equivalent, a nucleophile on an amino acid or nucleic acid base forms a covalent bond with formaldehyde, which is often stabilized in a second step involving another functional group on another molecule, leading to the formation of a methylene crosslink. When the crosslinking agent is an oxidizing agent, the oxidizing agent can react with the side chains of proteins and other biomolecules, enabling the formation of crosslinks that stabilize tissue structures.

[0056] As used herein, the term “primary antibody” means an antibody that directly binds to the antigen of interest. As used herein, the term “secondary antibody” means an antibody conjugated to a detection label. In some embodiments, the secondary antibody provided herein binds directly to the primary antibody. In other embodiments, the secondary antibody provided herein binds indirectly to the primary antibody, for example, by binding to another antibody that recognizes the primary antibody.

[0057] As used herein, the terms “fixation” or “to fix” refer to the process of fixing a biological specimen in an ISH (Integrated Stimulation) procedure, for example, to protect the biological specimen from disintegration due to autodegradation or decay. Fixation can also halt any ongoing biochemical reactions and improve the mechanical strength or stability of the treated tissue.

[0058] As used herein, the term “one or more” means, for example, one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twenty or more, thirty or more, forty or more, fifty or more, or a larger number as desired for a particular use.

[0059] As used herein, the term “detect” generally means any form of measurement, including the determination of whether or not a certain component is present. This term includes quantitative and / or qualitative determinations.

[0060] The terms “nucleic acid” and “polynucleotide” are used herein synonymously to refer to macromolecules of any length composed of nucleotides (e.g., deoxyribonucleotides or ribonucleotides), or synthetically produced compounds that can sequence-specifically hybridize with naturally occurring nucleic acids, similar to how two naturally occurring nucleic acids sequence-specifically hybridize, such as compounds that may be involved in Watson-Crick base-pair interactions. The term “multiple bases” (or “bases”), when used herein in relation to a polynucleotide sequence, is synonymous with “multiple nucleotides” (or “nucleotides”), i.e., monomer subunits of a polynucleotide. The terms “nucleoside” and “nucleotide” are intended to include not only the moieties containing known purine and pyrimidine bases, but also moieties containing other modified heterocyclic bases. Such modifications include methylated purines or methylated pyrimidines, acylated purines or acylated pyrimidines, alkylated riboses, or other heterocycles. Furthermore, the terms “nucleoside” and “nucleotide” encompass not only the conventional ribose and deoxyribose-containing portions but also portions containing other sugars. Modified nucleosides or nucleotides also include modifications to the sugar portion, for example, one or more hydroxyl groups being replaced with halogen atoms or aliphatic groups, or being functionalized with ethers or amines, etc. “Analog” refers to molecules that are mimetic, derivatives, have a similar structure, or have structural features recognized in the literature as other similar terms, including, for example, polynucleotides incorporating non-natural nucleotides, nucleotide mimetic (such as 2'-modified nucleosides), peptide nucleic acids, oligomeric nucleoside phosphonates, and any polynucleotide to which substituents (such as protecting groups or linking moieties) have been added.

[0061] The term "complementary" refers to a specific binding between polynucleotides based on their sequences. As used herein, a first polynucleotide and a second polynucleotide are complementary if they bind to each other in a hybridization assay under stringent conditions, for example, if they produce a signal at a predetermined or detectable level in the hybridization assay. The polynucleotide portions are complementary if they follow conventional base pairing rules (e.g., A pairs with T (or U), and G pairs with C), but small regions of mismatched, inserted, or deleted sequences (e.g., less than about 3 bases) may be present.

[0062] As used herein, the term "sample" refers to a substance or mixture of substances containing one or more components of the subject. The term "sample" encompasses "biological sample," which means a sample obtained from a biological subject, such as a sample derived from biological tissue or fluid obtained, reached, or collected in vivo or in situ. Biological samples also include samples derived from a region of a biological subject containing precancerous cells, cancer cells, precancerous tissue, or cancerous tissue. Such samples may, but are not limited to, organs, tissues, cells, and exosomes isolated from mammals. Exemplary biological samples include, but are not limited to, cell lysates, cells, cell cultures, cell lines, tissues, oral tissues, gastrointestinal tissues, organs, organoids, biological fluids, blood samples, urine samples, and skin samples. Preferred biological samples include, but are not limited to, whole blood, partially purified blood, PBMCs, and tissue biopsies.

[0063] As used herein, the term “probe” refers to a capture substance directed towards a specific target mRNA sequence. Thus, each probe in a set of probes has its own target mRNA sequence. In some embodiments, probes can be used individually. In other embodiments, probes can be used as part of a set. In some embodiments, the probes provided herein are “nucleic acid probes” or “oligonucleotide probes,” which refer to nucleic acids that have a complementary sequence and can typically bind to a target nucleic acid, such as the mRNA biomarker provided herein, through complementary base pairing by hydrogen bonding. As used herein, probes may contain native bases (e.g., A, G, C, or T) or modified bases (e.g., 7-deazaguanosine, inosine). In addition, bases within a probe may be linked by bonds other than phosphodiester bonds, provided that they do not interfere with hybridization. Probes can be labeled directly or indirectly with tags, such as chromophores, lumiphores, or plastids. The presence or absence of a target mRNA biomarker can be detected by assaying for the presence or absence of a probe.

[0064] As used in this disclosure and claims, the singular forms "a," "an," and "the" refer to multiple subjects unless the context explicitly indicates otherwise.

[0065] When an embodiment is described herein with the term “comprising,” it is understood that similar embodiments described in terms of “consisting of” and / or “consisting essentially of” are also provided. Similarly, when an embodiment is described herein with the expression “consisting essentially of,” it is understood that similar embodiments described in terms of “consisting essentially of” are also provided.

[0066] The term "between," as used in expressions such as "between A and B" or "between AB," indicates a range that includes both A and B.

[0067] In this specification, the term "and / or" as used in expressions such as "A and / or B" is intended to include A and B, A or B, A only, and B only. Similarly, the term "and / or" as used in expressions such as "A, B and / or C" is intended to include each of the following specific examples: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A only; B only; and C only.

[0068] 6.2 Integrated ISH / IHC Co-detection In one embodiment, the Specified provides a method for preparing a biological sample for the simultaneous detection of a target nucleic acid and a target protein, comprising: (i) incubating the biological sample with a primary antibody; (ii) treating the biological sample with a crosslinking agent after (i); and (iii) detecting the target nucleic acid by in situ hybridization after (ii).

[0069] In some embodiments, the method further includes treating the biological sample with a protease after treating it with a crosslinking agent and before detecting the target nucleic acid by in situ hybridization. In other embodiments, the method further includes incubating the biological sample with a secondary antibody or other labeling method after detecting the target nucleic acid by in situ hybridization.

[0070] In some embodiments, the preparation method provided herein includes sequentially performing the following steps: incubating a biological sample with a primary antibody; treating the biological sample with a crosslinking agent; treating the biological sample with a protease; detecting a target nucleic acid by ISH; and incubating the biological sample with a secondary antibody or other labeling method. In some embodiments, additional steps may be included.

[0071] Integrated Strain Spectroscopy (ISH) is a powerful technique for locating specific target nucleic acids within fixed tissues and cells, thereby obtaining temporal and spatial information about, for example, gene expression and gene loci. A labeled probe hybridizes to the target nucleic acid sequence within the sample. This labeled probe can then be detected. ISH involves preparing whole cell preparations, paraffin-embedded tissues, frozen tissues, or suspension sections. If the preparation is paraffin-embedded tissue, the next step in ISH is deparaffinization, which involves removing the paraffin and rehydrating the sample. In some embodiments, the ISH steps provided herein include a blocking step, in which a specific blocking agent(s) may be applied to block specific endogenous components of the cell, thereby reducing the assay background. For example, when horseradish peroxidase (HRP) is used as the detection enzyme in a later step, hydrogen peroxide is the blocking agent. Hydrogen peroxide is added to inactivate endogenous HRP activity in the sample, thereby reducing the assay background. In certain embodiments, this blocking step is added as a first pretreatment step immediately after deparaffinization. In some embodiments, the ISH step provided herein includes an epitope antigen retrieval step, to which a specific epitope antigen retrieval buffer(s) may be added to expose the target nucleic acid. In some embodiments, the epitope antigen retrieval step includes heating the sample. In some embodiments, the epitope antigen retrieval step includes heating the sample to about 50°C to about 100°C. In one embodiment, the epitope antigen retrieval step includes heating the sample to about 88°C. In some embodiments, the ISH provided herein includes a protease step. Surfactants (e.g., Triton X-100 or SDS) and proteinase K can be used to increase the permeability of the fixed cells. Typically, surfactant treatment using Triton X-100 or SDS is frequently used to extract lipids and permeate the cell membrane. Proteinase K is a non-specific protease that is active over a wide pH range and is not easily inactivated.Proteinase K is used to digest the proteins surrounding the target nucleic acid. The optimal concentration and duration of the treatment can be determined experimentally, as is well known in the art. In some embodiments, the ISH provided herein includes dehydrating the biological sample. In certain embodiments, dehydration is carried out by increasing the concentration of ethanol, for example, in the order of 70%, 95%, and 100% ethanol. In some embodiments, the ISH provided herein includes incubating the biological sample with a hybridization buffer. In certain embodiments, the hybridization buffer is formamide-based. In some embodiments, the ISH provided herein includes incubating the biological sample with a hybridization probe(s). In some embodiments, the ISH provided herein includes amplifying and detecting the hybridization probe(s).

[0072] Like ISH, IHC is another widely used technique in that it can preserve temporal and spatial information, but it differs in that it is for detecting target proteins. Leveraging the advantage of antigen-antibody specificity, IHC allows for the visualization and documentation of highly identifiable distribution and localization of specific target proteins within cells and within their appropriate histological backgrounds. IHC involves preparing whole cell preparations, paraffin-embedded tissues, frozen tissues, or suspension sections. If the preparation is paraffin-embedded tissue, the next step in IHC is deparaffinization, which involves removing the paraffin. If crosslinking agents, such as formalin, are used during the tissue preparation and preservation steps, formalin fixation may mask epitopes and lead to reduced immunoactivity (see Arnold et al., Biotech Histochem 71:224-230 (1996)). Formalin fixation is a time-dependent process in which, with increasing fixation time, the formaldehyde group continues to bind to the protein until it reaches an equilibrium point (see Fox et al., J Histochem Cytochem 33:845-853 (1985)). Studies have shown that formalin fixation, especially prolonged formalin fixation, leads to a reduction in antigenicity (see Battifora and Kopinski, J Histochem Cytochem 34:1095-1100 (1986)), which has limited the use of formalin-fixed tissues in diagnostic IHC (see Ramos-Vara, Vet Pathol 42:405-426 (2005), Webster et al., J Histochem Cytochem. 57(8):753-761 (2009)).

[0073] The benefits of simultaneous detection of target nucleic acids using techniques such as ISH and target proteins using techniques such as IHC are enormous. For example, simultaneous detection increases the processing capacity of the analysis and reduces the time and cost burden associated with investigating their individual components. Furthermore, by combining detections in the same sample, researchers can obtain information that cannot be obtained from staining separate sections, such as visualizing secreted proteins and the cell(s) from which they originate, or information on interactions between different cell types. Although the steps of both techniques appear similar, there are problems in combining the two techniques in a single sample.

[0074] Theoretically, the obvious choice for combining ISH and IHC is to perform IHC first, followed by ISH (see, e.g., Kochan et al., BioTechniques, 59(4):209-221 (2018)). Although there have been reports of successful combinations of immunofluorescence and RNA FISH, these have been unsatisfactory in terms of signal intensity, staining pattern, and reagent compatibility, leading to artificial results and failures in experiments. Furthermore, IHC performed in the past may have needed to be RNase-free, which is impractical for larger-scale or broader applications because RNases contaminate many standard laboratory reagents unless extremely careful attention is paid.

[0075] Alternatively, simultaneous detection of target proteins and target nucleic acids on the same tissue section may be achieved by first performing ISH to preserve mRNA integrity, followed immediately by IHC (see, e.g., Stempl et al., Molecular Vision 20, 1366-1373 (2014), Grabinski et al., PLoS ONE 10(3):e0120120 (2015)). However, there are several disadvantages to this protocol. Firstly, this method is often not compatible with all target antibodies. Secondly, the IHC signal performed later is frequently lost or significantly diminished by the early steps of this method. One reason for this is the harsh reagents and conditions used at the beginning of ISH, which result in substandard material for subsequent downstream assays on the same tissue section. For example, ISH pretreatment uses proteases to aid reagent penetration and facilitate access to the target. This protease step can, in some cases, damage the epitope, thereby impairing antibody-antigen binding and resulting in a decrease in IHC signaling. In addition, the formamide-based buffers and high-temperature incubation commonly used in ISH can impair non-covalent antibody-antigen binding, thereby reducing IHC signaling.

[0076] The method provided herein overcomes the above-mentioned problems by modifying conventional protocols, for example, by stabilizing antigen-antibody binding. This stabilization is achieved, for example, by first incubating the sample with the primary antibody and fixing the sample with 10% NBF before ISH detection, followed by the remaining IHC staining.

[0077] In some specific embodiments, the method provided herein comprises crosslinking the sample after binding of the primary antibody, followed by protease treatment and ISH, and then the remaining IHC staining steps. This method has the ability to both protect the epitope from protease activity and stabilize antigen-antibody binding through subsequent steps involving hybridization buffer-dependent amplification. This method provides high flexibility in co-detection of proteins and nucleic acids, maintains highly discriminable detection of nucleic acids, and stabilizes the IHC signal of antibodies that were previously unsuitable for conventional co-detection.

[0078] As illustrated in sections 7.2 and 7.3 below, the methods provided herein enable the detection of antibodies, such as CD20, that were previously considered unsuitable for conventional co-detection by ISH and IHC. Conventionally, when ISH is performed first to preserve the signal from the target nucleic acid, the IHC signal may be impaired. For example, if the same sample is treated with an ISH procedure, such as protease treatment, the epitope may be negatively affected. Therefore, it is beneficial to incubate the sample with the antibody before any potentially problematic ISH procedure. Furthermore, as illustrated in Figures 6C-6E, a crosslinking step is added after incubation of the primary antibody for ISH and IHC integration. Crosslinking improves antigen-antibody binding, enabling the detection and visualization of protein epitopes that were previously unsuitable for ISH conditions. In addition, the crosslinking step provided herein has been shown to improve the IHC signal by protecting antibody-antigen binding in the sample from protease treatment and incubation with hybridization buffer (see Figures 3A-3C and 4A-4C).

[0079] Furthermore, the methods provided herein have shown additional boosting effects for several target antibodies. For example, crosslinking before incubation and / or protease treatment of the hybridization buffer improved the IHC signal of the CD8 antibody, not only compared to the conventional ISH-IHC protocol but also compared to the standard IHC protocol alone (see Figures 4A-4F and 5A-5D). Similar boosting effects in IHC detection apply to additional antibodies. In some embodiments, similar procedures, such as an additional crosslinking step after primary antibody incubation, may be used to enhance the IHC signal as an attempt to detect a target protein alone. In other embodiments, protease treatment may be used to enhance the IHC signal as an attempt to detect a target protein alone. In other embodiments, procedures such as an additional crosslinking step after primary antibody incubation and experimental steps that mimic some or all of the ISH procedure may be used to enhance the IHC signal as an attempt to detect a target protein alone.

[0080] Several pretreatment steps may be required before hybridization using ISH probes. For example, when specific blocking agents are applied, ISH blocking is intended to block specific endogenous components of cells, thereby reducing the assay background. For example, when horseradish peroxidase (HRP) is used as the detection enzyme in a later step, hydrogen peroxide is the blocking agent. Adding hydrogen peroxide inactivates endogenous HRP activity in the sample, thereby reducing the assay background. In certain embodiments, this ISH blocking is added as the first pretreatment step immediately after deparaffinization. In some embodiments, the pretreatment step includes a target antigen retrieval step, in which specific target antigen retrieval buffers may be added to expose the target nucleic acid. In some embodiments, the epitope antigen retrieval step includes heating the sample. In some embodiments, the target antigen retrieval step includes heating the sample to about 50°C to about 100°C. In one embodiment, the target antigen retrieval step includes heating the sample to approximately 88°C. In some embodiments, the pretreatment step includes protease treatment, which is used to digest the proteins surrounding the target nucleic acid. The optimal concentration and duration of the treatment can be determined experimentally, as is well known in the art. In certain embodiments, the protease treatment is carried out using trypsin. In certain embodiments, the protease treatment is carried out using proteinase K. In certain embodiments, the protease treatment is carried out using pepsin. In certain embodiments, the protease treatment is carried out using pronase. In certain embodiments, the protease treatment is carried out using endoproteinase AspN. In certain embodiments, the protease treatment is carried out using endoproteinase GluC.

[0081] In some embodiments, the method for preparing a biological sample for the simultaneous detection of target nucleic acids and target proteins provided herein includes incubation of the biological sample with a primary antibody, followed by a first treatment of the biological sample with a crosslinking agent, then detection of the target nucleic acid by ISH, and the remaining steps of IHC. In other embodiments, the method provided herein includes incubation of the biological sample with a primary antibody, followed by a first treatment of the biological sample with a crosslinking agent, then protease treatment, then detection of the target nucleic acid by ISH, and the remaining steps of IHC, as described in the preceding paragraph. In other embodiments, the method provided herein includes incubation of the biological sample with a primary antibody, followed by a first treatment of the biological sample with a crosslinking agent, then protease treatment, then ISH blocking, then detection of the target nucleic acid by ISH, and the remaining steps of IHC, as described in the preceding paragraph. In other embodiments, the method provided herein includes incubation of the biological sample with a primary antibody, followed by a first treatment of the biological sample with a crosslinking agent, then ISH blocking, then protease treatment, then detection of the target nucleic acid by ISH, and the remaining steps of IHC, as described in the preceding paragraph. In other embodiments, the method provided herein includes incubating a biological sample with a primary antibody, followed by a first treatment of the biological sample with a crosslinking agent, then ISH blocking, then ISH detection of the target nucleic acid, and the remaining steps of IHC, as described in the preceding paragraph. In other embodiments, the method provided herein includes target antigen retrieval, followed by incubating a biological sample with a primary antibody, followed by a first treatment of the biological sample with a crosslinking agent, then ISH detection of the target nucleic acid, and the remaining steps of IHC, as described in the preceding paragraph. In other embodiments, the method provided herein includes target antigen retrieval, followed by incubating a biological sample with a primary antibody, followed by a first treatment of the biological sample with a crosslinking agent, then protease treatment, then ISH detection of the target nucleic acid, and the remaining steps of IHC, as described in the preceding paragraph.In other embodiments, the method provided herein includes the steps of target antigen retrieval, incubation of the biological sample with a primary antibody, followed by a first treatment of the biological sample with a crosslinking agent, followed by protease treatment, followed by ISH blocking, followed by ISH detection of the target nucleic acid, and the remainder of IHC, as described in the preceding paragraph. In other embodiments, the method provided herein includes incubating a biological sample with a primary antibody, followed by a first treatment of the biological sample with a crosslinking agent, then antigen retrieval of the target, then detection of the target nucleic acid by ISH, and the remaining steps of IHC, as described in the preceding paragraph. In other embodiments, the method provided herein includes incubating a biological sample with a primary antibody, followed by a first treatment of the biological sample with a crosslinking agent, then antigen retrieval of the target, then protease treatment, then detection of the target nucleic acid by ISH, and the remaining steps of IHC, as described in the preceding paragraph. In other embodiments, the method provided herein includes incubating a biological sample with a primary antibody, followed by a first treatment of the biological sample with a crosslinking agent, then antigen retrieval of the target, then protease treatment, then ISH blocking, then detection of the target nucleic acid by ISH, and the remaining steps of IHC, as described in the preceding paragraph.In other embodiments, the method provided herein includes incubating a biological sample with a primary antibody, followed by a first treatment of the biological sample with a crosslinking agent, then target antigen retrieval, then ISH blocking, then protease treatment, then ISH detection of the target nucleic acid, and the remaining steps of IHC, as described in the preceding paragraph. In other embodiments, the method provided herein includes incubating a biological sample with a primary antibody, followed by a first treatment of the biological sample with a crosslinking agent, then target antigen retrieval, then ISH blocking, then ISH detection of the target nucleic acid, and the remaining steps of IHC, as described in the preceding paragraph. In other embodiments, the method provided herein includes the use of three or more antibodies, where a secondary antibody or other subsequent antibody is applied after the primary antibody, and the secondary antibody or other detection method is applied after ISH.

[0082] In some embodiments, the methods provided herein involve treating a biological sample with a crosslinking agent. In certain embodiments, the crosslinking agent is a fixative. In certain embodiments, the crosslinking agent is formaldehyde. In certain embodiments, the crosslinking agent is glutaraldehyde. In certain embodiments, the crosslinking agent is acrolein. In certain embodiments, the crosslinking agent is osmium tetroxide. In certain embodiments, the crosslinking agent is a type of permanganate fixative. In one embodiment, the crosslinking agent is potassium permanganate. In certain embodiments, the crosslinking agent is a type of dichromate fixative. In one embodiment, the crosslinking agent is potassium dichromate. In certain embodiments, the crosslinking agent is chromic acid.

[0083] In some embodiments, the methods provided herein involve treating a biological sample with a crosslinking agent, which is a mixture of crosslinkable fixatives. In some embodiments, the crosslinking agent is a mixed solution of two or more fixatives selected from the list of formaldehyde, glutaraldehyde, acrolein, osmium tetroxide, permanganate fixatives, dichromate fixatives, and chromic acid. In one embodiment, the crosslinking agent is Bouin fixative, which is a solution of picric acid, formaldehyde, and acetic acid. In one embodiment, the crosslinking agent is a mixture of formaldehyde and glutaraldehyde. In one embodiment, the crosslinking agent is FAA, which is a solution of ethanol, acetic acid, and formaldehyde. In one embodiment, the crosslinking agent is periodate-lysine-paraformaldehyde (PLP), which is a solution of paraformaldehyde, L-lysine, and INaO4. In one embodiment, the crosslinking agent is phosphate-buffered formalin (PBF). In one embodiment, the crosslinking agent is formal calcium, which is a solution of formaldehyde and calcium chloride. In one embodiment, the crosslinking agent is formal saline solution, which is a solution of formaldehyde and sodium chloride. In one embodiment, the crosslinking agent is zinc formalin, which is a solution of formaldehyde and zinc sulfate. In one embodiment, the crosslinking agent is Helly fixative, which is a solution of formaldehyde, potassium dichromate, sodium sulfate, and mercury chloride. In one embodiment, the crosslinking agent is Hollande fixative, which is a solution of formaldehyde, copper acetate, picric acid, and acetic acid. In one embodiment, the crosslinking agent is Gendre solution, which is a solution of formaldehyde, ethanol, picric acid, and glacial acetic acid. In one embodiment, the crosslinking agent is alcohol formalin, which is a solution of formaldehyde, ethanol, and calcium acetate. In one embodiment, the crosslinking agent is formal acetate alcohol, which is a solution of formaldehyde, glacial acetic acid, and ethanol. In one embodiment, the crosslinking agent is a mixture of fixatives, at least one of which is formaldehyde or glutaraldehyde. In one embodiment, the crosslinking agent is a fixative that is not used simultaneously but is used separately or sequentially, and at least one of the fixatives is formaldehyde or glutaraldehyde.

[0084] In some embodiments, the methods provided herein involve treating a biological sample with two or more crosslinking agents, which are not applied simultaneously but separately or sequentially, and are selected from the list of formaldehyde, glutaraldehyde, acrolein, osmium tetroxide, permanganate fixatives, dichromate fixatives, and chromic acid.

[0085] In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 0°C to about 100°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 1°C to about 90°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 2°C to about 80°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 3°C ​​to about 70°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 4°C to about 60°C.

[0086] In some embodiments, the methods provided herein include treating a biological sample with about 1% to about 20% neutral buffered formalin (NBF) at a temperature of about 0°C to about 100°C. In some embodiments, the methods provided herein include treating a biological sample with about 1% to about 20% NBF at a temperature of about 1°C to about 90°C. In some embodiments, the methods provided herein include treating a biological sample with about 1% to about 20% NBF at a temperature of about 2°C to about 80°C. In some embodiments, the methods provided herein include treating a biological sample with about 1% to about 20% NBF at a temperature of about 3°C ​​to about 70°C. In some embodiments, the methods provided herein include treating a biological sample with about 1% to about 20% NBF at a temperature of about 4°C to about 60°C.

[0087] In some embodiments, the method provided herein includes treating a biological sample with about 10% NBF at a temperature of about 0°C to about 100°C. In some embodiments, the method provided herein includes treating a biological sample with about 10% NBF at a temperature of about 1°C to about 90°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 2°C to about 80°C. In some embodiments, the method provided herein includes treating a biological sample with about 10% NBF at a temperature of about 3°C ​​to about 70°C. In some embodiments, the method provided herein includes treating a biological sample with about 10% NBF at a temperature of about 4°C to about 60°C.

[0088] In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 1°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 2°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 3°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 4°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 5°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 6°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 7°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 8°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 9°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 10°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 11°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 12°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 13°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 14°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 15°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 16°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 17°C.In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 18°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 19°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 20°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 21°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 22°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 23°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 24°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 25°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 26°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 27°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 28°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 29°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 30°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 35°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 40°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 45°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 50°C.In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 55°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 60°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 65°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 70°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 75°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 80°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 85°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 90°C. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of about 95°C. In some embodiments, the methods provided herein include treating a biological sample with a crosslinking agent at a temperature of about 100°C.

[0089] In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 1°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 2°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 3°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 4°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 5°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 6°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 7°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 8°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 9°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 10°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 11°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 12°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 13°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 14°C. In some embodiments, the methods provided herein include treating a biological sample with 1% to 20% NBF at a temperature of about 15°C.In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 16°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 17°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 18°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 19°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 20°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 21°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 22°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 23°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 24°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 25°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 26°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 27°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 28°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 29°C. In some embodiments, the methods provided herein include treating a biological sample with 1% to 20% NBF at a temperature of about 30°C.In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 35°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 40°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 45°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 50°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 55°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 60°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 65°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 70°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 75°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 80°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 85°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 90°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 95°C. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF at a temperature of about 100°C.

[0090] In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 1°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 2°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 3°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 4°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 5°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 6°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 7°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 8°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 9°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 10°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 11°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 12°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 13°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 14°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 15°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 16°C.In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 17°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 18°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 19°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 20°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 21°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 22°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 23°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 24°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 25°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 26°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 27°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 28°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 29°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 30°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 35°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 40°C.In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 45°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 50°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 55°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 60°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 65°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 70°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 75°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 80°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 85°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 90°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 95°C. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF at a temperature of about 100°C.

[0091] In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 0.1 hours to about 48 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 0.1 hours to about 36 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 0.1 hours to about 24 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 0.2 hours to about 22 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 0.25 hours to about 20 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 0.25 hours to about 18 hours.

[0092] In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 0.1 hours to about 48 hours. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 0.1 hours to about 36 hours. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 0.1 hours to about 24 hours. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 0.2 hours to about 22 hours. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 0.25 hours to about 20 hours. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 0.25 hours to about 18 hours.

[0093] In some embodiments, the method provided herein includes treating a biological sample with 10% NBF for about 0.1 hours to about 48 hours. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF for about 0.1 hours to about 36 hours. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF for about 0.1 hours to about 24 hours. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF for about 0.2 hours to about 22 hours. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF for about 0.25 hours to about 20 hours. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF for about 0.25 hours to about 18 hours.

[0094] In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 0.1 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 0.25 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 0.5 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 0.75 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 1 hour. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 2 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 3 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 4 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 5 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 6 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 7 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 8 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 9 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 10 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 11 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 12 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 14 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 18 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 20 hours.In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 24 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 36 hours. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent for about 48 hours.

[0095] In some embodiments, the method provided herein includes treating the biological sample with 1% to 20% NBF for about 0.1 hours. In some embodiments, the method provided herein includes treating the biological sample with 1% to 20% NBF for about 0.25 hours. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 0.5 hours. In some embodiments, the method provided herein includes treating the biological sample with 1% to 20% NBF for about 0.75 hours. In some embodiments, the method provided herein includes treating the biological sample with 1% to 20% NBF for about 1 hour. In some embodiments, the method provided herein includes treating the biological sample with 1% to 20% NBF for about 2 hours. In some embodiments, the method provided herein includes treating the biological sample with 1% to 20% NBF for about 3 hours. In some embodiments, the method provided herein includes treating the biological sample with 1% to 20% NBF for about 4 hours. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 5 hours. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 6 hours. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 7 hours. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 8 hours. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 9 hours. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 10 hours. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 11 hours. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 12 hours. In some embodiments, the methods provided herein include treating a biological sample with 1% to 20% NBF for about 14 hours.In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 18 hours. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 20 hours. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 24 hours. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 36 hours. In some embodiments, the method provided herein includes treating a biological sample with 1% to 20% NBF for about 48 hours.

[0096] In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 0.1 hours. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 0.25 hours. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 0.5 hours. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 0.75 hours. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 1 hour. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 2 hours. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 3 hours. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 4 hours. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 5 hours. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 6 hours. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 7 hours. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 8 hours. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 9 hours. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 10 hours. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 11 hours. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 12 hours. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 14 hours. In some embodiments, the method provided herein includes treating the biological sample with 10% NBF for about 18 hours.In some embodiments, the method provided herein includes treating a biological sample with 10% NBF for about 20 hours. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF for about 24 hours. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF for about 36 hours. In some embodiments, the method provided herein includes treating a biological sample with 10% NBF for about 48 hours.

[0097] In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 4°C for more than 10 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 4°C for more than 5 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 4°C for more than 1 hour. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 4°C for about 5 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 4°C for about 6 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 4°C for about 7 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 4°C for about 8 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 4°C for about 9 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 4°C for about 10 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 4°C for about 11 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 4°C for about 12 hours.

[0098] In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for less than about 6 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for less than about 3 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for less than about 1 hour. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for less than about 0.5 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 0.1 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 0.15 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 0.2 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 0.25 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 0.3 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 0.35 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 0.4 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 0.45 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 0.5 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 0.55 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 0.6 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 0.65 hours.In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 0.7 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 0.75 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 0.8 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 0.85 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 0.9 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 1 hour. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 1.5 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 2 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 2.5 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 3 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 3.5 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at room temperature for about 4 hours.

[0099] In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 40°C for less than 6 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 40°C for less than 3 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 40°C for less than 1 hour. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 40°C for less than 0.5 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 40°C for about 0.1 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 40°C for about 0.25 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 40°C for about 0.5 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 40°C for about 0.75 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 40°C for about 1 hour.

[0100] In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 60°C for less than about 6 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 60°C for less than about 3 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 60°C for less than about 1 hour. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 60°C for less than about 0.5 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 60°C for about 0.1 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 60°C for about 0.25 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 60°C for about 0.5 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 60°C for about 0.75 hours. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a temperature of 60°C for about 1 hour.

[0101] In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a pH of about 6. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a pH of about 6.5. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a pH of about 7. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a pH of about 7.5. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a pH of about 8. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a pH of about 8.5. In some embodiments, the method provided herein includes treating a biological sample with a crosslinking agent at a pH of about 9.

[0102] In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of less than 1%. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of more than 50%. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of 1% to 50%. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of 2% to 40%. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of 3% to 30%. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of 4% to 20%. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of about 4%. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of 12%. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of 24%. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of 37%.

[0103] In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of formaldehyde or equivalent at 1% to 50%. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of formaldehyde or equivalent at 2% to 40%. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of formaldehyde or equivalent at 3% to 30%. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of formaldehyde or equivalent at 4% to 20%. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of formaldehyde or equivalent at about 4%. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of formaldehyde or equivalent at 12%. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of formaldehyde or equivalent at 24%. In some specific embodiments, the method provided herein includes treating a biological sample with a crosslinking agent having an effective concentration of formaldehyde or equivalent at 37%.

[0104] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 6 and an effective concentration of less than 12% formaldehyde or equivalent, where the temperature is 4°C. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0105] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 7 and an effective concentration of less than 12% formaldehyde or equivalent, where the temperature is 4°C. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0106] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 8 and an effective concentration of less than 12% formaldehyde or equivalent, where the temperature is 4°C. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0107] In certain embodiments, the method provided herein involves treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 6 and an effective concentration of less than 12% formaldehyde or equivalent, where the temperature is room temperature. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0108] In certain embodiments, the method provided herein involves treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 7 and an effective concentration of less than 12% formaldehyde or equivalent, where the temperature is room temperature. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0109] In certain embodiments, the method provided herein involves treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 8 and an effective concentration of less than 12% formaldehyde or equivalent, where the temperature is room temperature. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0110] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 6 and an effective concentration of less than 12% formaldehyde or equivalent, where the temperature is 40°C. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0111] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 7 and an effective concentration of less than 12% formaldehyde or equivalent, where the temperature is 40°C. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0112] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 8 and an effective concentration of less than 12% formaldehyde or equivalent, where the temperature is 40°C. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0113] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 6 and an effective concentration of less than 12% formaldehyde or equivalent, where the temperature is 60°C. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0114] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 7 and an effective concentration of less than 12% formaldehyde or equivalent, where the temperature is 60°C. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0115] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 8 and an effective concentration of less than 12% formaldehyde or equivalent, where the temperature is 60°C. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0116] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 6 and an effective concentration of formaldehyde or equivalent greater than 12%, where the temperature is 4°C. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0117] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 7 and an effective concentration of formaldehyde or equivalent greater than 12%, where the temperature is 4°C. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0118] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of approximately 8 and an effective concentration of formaldehyde or equivalent greater than 12%, where the temperature is 4°C. In one embodiment, crosslinking lasts for less than approximately 0.5 hours. In one embodiment, crosslinking lasts for less than approximately 1 hour. In one embodiment, crosslinking lasts for more than approximately 1 hour. In one embodiment, crosslinking lasts for more than approximately 6 hours. In one embodiment, crosslinking lasts for more than approximately 12 hours. In one embodiment, crosslinking lasts for more than approximately 18 hours. In one embodiment, crosslinking lasts for more than approximately 24 hours. In one embodiment, crosslinking lasts for more than approximately 30 hours. In one embodiment, crosslinking lasts for more than approximately 36 hours. In one embodiment, crosslinking lasts for more than approximately 48 hours.

[0119] In certain embodiments, the method provided herein involves treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 6 and an effective concentration of formaldehyde or equivalent greater than 12%, where the temperature is room temperature. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0120] In certain embodiments, the method provided herein involves treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 7 and an effective concentration of formaldehyde or equivalent greater than 12%, where the temperature is room temperature. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0121] In certain embodiments, the method provided herein involves treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 8 and an effective concentration of formaldehyde or equivalent greater than 12%, where the temperature is room temperature. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0122] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 6 and an effective concentration of formaldehyde or equivalent greater than 12%, where the temperature is 40°C. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0123] In certain embodiments, the method provided herein involves treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of approximately 7 and an effective concentration of formaldehyde or equivalent greater than 12%, where the temperature is 40°C. In one embodiment, crosslinking lasts for less than approximately 0.5 hours. In one embodiment, crosslinking lasts for less than approximately 1 hour. In one embodiment, crosslinking lasts for more than approximately 1 hour. In one embodiment, crosslinking lasts for more than approximately 6 hours. In one embodiment, crosslinking lasts for more than approximately 12 hours. In one embodiment, crosslinking lasts for more than approximately 18 hours. In one embodiment, crosslinking lasts for more than approximately 24 hours. In one embodiment, crosslinking lasts for more than approximately 30 hours. In one embodiment, crosslinking lasts for more than approximately 36 hours. In one embodiment, crosslinking lasts for more than approximately 48 hours.

[0124] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of approximately 8 and an effective concentration of formaldehyde or equivalent greater than 12%, where the temperature is 40°C. In one embodiment, crosslinking lasts for less than approximately 0.5 hours. In one embodiment, crosslinking lasts for less than approximately 1 hour. In one embodiment, crosslinking lasts for more than approximately 1 hour. In one embodiment, crosslinking lasts for more than approximately 6 hours. In one embodiment, crosslinking lasts for more than approximately 12 hours. In one embodiment, crosslinking lasts for more than approximately 18 hours. In one embodiment, crosslinking lasts for more than approximately 24 hours. In one embodiment, crosslinking lasts for more than approximately 30 hours. In one embodiment, crosslinking lasts for more than approximately 36 hours. In one embodiment, crosslinking lasts for more than approximately 48 hours.

[0125] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 6 and an effective concentration of formaldehyde or equivalent greater than 12%, where the temperature is 60°C. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0126] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 7 and an effective concentration of formaldehyde or equivalent greater than 12%, where the temperature is 60°C. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0127] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 8 and an effective concentration of formaldehyde or equivalent greater than 12%, where the temperature is 60°C. In one embodiment, crosslinking lasts for less than about 0.5 hours. In one embodiment, crosslinking lasts for less than about 1 hour. In one embodiment, crosslinking lasts for more than about 1 hour. In one embodiment, crosslinking lasts for more than about 6 hours. In one embodiment, crosslinking lasts for more than about 12 hours. In one embodiment, crosslinking lasts for more than about 18 hours. In one embodiment, crosslinking lasts for more than about 24 hours. In one embodiment, crosslinking lasts for more than about 30 hours. In one embodiment, crosslinking lasts for more than about 36 hours. In one embodiment, crosslinking lasts for more than about 48 hours.

[0128] In certain embodiments, the method provided herein comprises treating a biological sample with a crosslinking agent in an isotonic buffer having a pH of about 6 and an effective concentration of formaldehyde or equivalent greater than 12%, where the temperature is 4°C. In one embodiment, crosslinking lasts for about 1 hour or more. In one embodiment, crosslinking lasts for about 6 hours or more. In one embodiment, crosslinking lasts for about 12 hours or more. In one embodiment, crosslinking lasts for about 18 hours or more. In one embodiment, crosslinking lasts for about 24 hours or more. In one embodiment, crosslinking lasts for about 30 hours or more. In one embodiment, crosslinking lasts for about 36 hours or more. In one embodiment, crosslinking lasts for about 48 hours or more.

[0129] The methods provided herein include detecting a target nucleic acid. In some embodiments, the target nucleic acid is DNA. In other embodiments, the target nucleic acid is RNA.

[0130] In some embodiments, the methods provided herein are for preparing a biological sample for the simultaneous detection of target RNA and target protein, and include (i) incubation the biological sample with a primary antibody, (ii) treatment of the biological sample with a crosslinking agent after (i), and (iii) detection of the target RNA after (ii) by a hybridization-based method. In one embodiment, the method includes detecting the target RNA by ISH. In one embodiment, the method includes detecting the target RNA by a molecular beacon (see Tyagi et al., Nat Biotechnol. 4(3):303-308 (1996)). In one embodiment, the method includes detecting the target RNA by a forced intercalation (FIT) probe (see Kohler et al., Chembiochem. 6(1):69-77 (2005)). In some embodiments, the methods provided herein are for preparing a biological sample for the simultaneous detection of target RNA and target protein, and include (i) incubation the biological sample with a primary antibody, (ii) treatment of the biological sample with a crosslinking agent after (i), and (iii) detection of the target RNA by an aptamer-based method after (ii). In one embodiment, the method includes detecting the target RNA by the aptamer "Spinach" (see Paige et al., Science, 333(6042):642-646 (2011)). In some embodiments, the methods provided herein are for preparing a biological sample for the simultaneous detection of target RNA and target protein, and include (i) incubation the biological sample with a primary antibody, (ii) treatment of the biological sample with a crosslinking agent after (i), and (iii) detection of the target RNA by a particle-associated hybridization-based probe after (ii). In one embodiment, the method includes detecting the target RNA by a gold nanoparticle quantum dot probe.In some embodiments, the methods provided herein are for preparing a biological sample for the simultaneous detection of target RNA and target protein, and include (i) incubating the biological sample with a primary antibody, (ii) treating the biological sample with a crosslinking agent after (i), and (iii) detecting the target RNA by directly incorporating a visualizeable portion into the target RNA (see Jao et al., Proc Natl Acad Sci. 105(41):15779-15784 (2008)). In some embodiments, the methods provided herein are for preparing a biological sample for the simultaneous detection of target RNA and target protein, and include (i) incubating the biological sample with a primary antibody, (ii) treating the biological sample with a crosslinking agent after (i), and (iii) detecting the target RNA with an RNA-binding protein. In one embodiment, the method includes detecting the target RNA by a fluorescent protein fusion version of the bacteriophage MS2 coat protein (MCP) (see Park et al., Science. 343(6169):422-424 (2014)). In another embodiment, the method includes detecting the target RNA by a Pumilio-based method (see Kellermann et al., Chembiochem. 14(2):200-204 (2013)).

[0131] In some embodiments, the methods provided herein are for preparing a biological sample for the simultaneous detection of target DNA and target protein, and include (i) incubation the biological sample with a primary antibody, (ii) treatment of the biological sample with a crosslinking agent after (i), and (iii) detection of the target DNA after (ii) by a hybridization-based method. In some specific embodiments, the method includes detecting the target DNA by chromogenic ISH. In other specific embodiments, the method includes detecting the target DNA by fluorescent ISH.

[0132] In some embodiments, the method for preparing a biological sample for the simultaneous detection of target nucleic acids and target proteins includes biological samples of various origins. In one embodiment, the biological sample is a tissue sample or derived from a tissue sample. In one embodiment, the biological sample is a blood sample or derived from a blood sample. In one embodiment, the biological sample is a cytological sample or derived from a cytological sample. In one embodiment, the biological sample is cultured cells. In another embodiment, the biological sample is an exosome-containing sample.

[0133] Examples of tissue samples include tissue biopsy specimens. Examples of blood samples include blood samples collected for diagnostic purposes. In the case of blood samples, the blood can be analyzed directly, as in blood smears, or the blood can be processed, for example, by lysing erythrocytes, isolating PBMCs or leukocytes, or isolating target cells, so that the cells in the sample to be analyzed by the method of this disclosure are present in or extracted from the blood sample. Similarly, tissue samples can be processed, for example, by cutting the tissue sample into small pieces and processing it physically or enzymatically to destroy the tissue and bring it into the state of individual cells or cell clusters. In addition, if desired, cytological samples can be processed to isolate cells or destroy cell clusters. Thus, tissue, blood, and cytological samples can be obtained and processed using methods well known in the art. The method of this disclosure can be used for diagnostic purposes to confirm the presence or absence of diseased cells based on the presence or absence of nucleic acid targets, which are biomarkers indicating a pathological condition.

[0134] Those skilled in the art will understand that any number of suitable sample types can be used when detecting target nucleic acids and target proteins using the methods provided herein. The samples used in the methods provided herein are generally biological or tissue samples. Such samples can be obtained from living subjects and include samples derived from biological tissue or bodily fluids taken from an individual or any other source of biological material (such as a biological specimen, autopsy specimen, or forensic material). Biological samples also include samples obtained from areas of living subjects that contain, or are suspected to contain, precancerous cells, cancer cells, precancerous tissue, or cancerous tissue, such as tissue biopsies (including fine-needle aspirations, blood samples, or cytological specimens). Such samples may, but are not limited to, organs, tissues, tissue fragments, cells, and / or exosomes isolated from organisms such as mammals. Exemplary biological samples include, but are not limited to, cells, primary cell cultures, cell lines, tissues, organs, organoids, and cell cultures such as bodily fluids. Additional biological samples include, but are not limited to, skin samples, tissue biopsies (including fine-needle aspirations), cytological samples, stool samples, and bodily fluids (including blood and / or serum samples, saliva, and semen). Such samples can be used for medical or veterinary diagnostic purposes.

[0135] The method of obtaining cytological samples for analysis using the methods provided herein is well known in the art (see, for example, Dey, “Cytology Sample Procurement, Fixation and Processing” in Basic and Advanced Laboratory Techniques in Histopathology and Cytology pp.121-132, Springer, Singapore (2018), and “Non-Gynecological Cytology Practice Guideline” American Society of Cytopathology, Adopted by the ASC executive board March 2, 2004).

[0136] For example, methods for processing samples (including biopsy and cytological samples) for the analysis of cervical tissue are well known in the art (e.g., Cecil Textbook of Medicine, Bennett and Plum, eds., 20th ed., WB Saunders, Philadelphia (1996), Colposcopy and Treatment of Cervical Intraepithelial Neoplasia: A Beginner's Manual, Sellors and Sankaranarayanan, eds., International Agency for Research on Cancer, Lyon, France (2003), Kalaf and Cooper, J. Clin. Pathol. 60:449-455 (2007), Brown and Trimble, Best Pract. Res. Clin. Obstet. Gynaecol. 26:233-242 (2012), Waxman et al., Obstet. Gynecol.120:1465-1471 (2012), Cervical Cytology Practice Guidelines TOC, Approved by the American Society of Cytopathology (ASC) Executive Board, November 10, 2000).

[0137] In certain embodiments, the sample is a tissue sample or derived from a tissue sample. In some embodiments, the tissue sample is FFPE. In some embodiments, the tissue sample is fresh-frozen. In some embodiments, the tissue sample is prepared with a fixative. In some embodiments, the tissue sample is prepared with a crosslinking fixative. In other specific embodiments, the sample is a blood sample or derived from a blood sample. In yet another specific embodiment, the sample is a cytological sample or derived from a cytological sample.

[0138] 6.3 Method for simultaneously detecting target nucleic acids and target proteins In another embodiment, this specification provides a method for simultaneously detecting a target nucleic acid and a target protein in a biological sample, comprising: (i) incubating the biological sample with a primary antibody; (ii) treating the biological sample with a crosslinking agent; (iii) treating the biological sample with a protease; (iv) detecting the target nucleic acid by in situ hybridization; and (v) detecting the target protein by incubating the biological sample with a secondary antibody or other labeling method.

[0139] In addition to what is described in Section 6.2 above, ISH includes hybridizing a target nucleic acid with one or more target probes. Methods for in situ detection of nucleic acids are well known to those skilled in the art (see, for example, US2008 / 0038725, US2009 / 0081688, Hicks et al., J.Mol.Histol. 35:595-601 (2004)). As used herein, “in situ hybridization” or “ISH” means a type of hybridization in which a directly or indirectly labeled complementary DNA or RNA strand (such as a probe) is bound to a given nucleic acid in a sample, specifically a tissue or cell part or section (in situ), to locate that nucleic acid. The types of probes may be double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded complementary RNA (sscRNA), and / or synthetic oligonucleotides.

[0140] In some embodiments, the ISH provided herein includes providing at least one set of one or more target probes capable of hybridizing to the target nucleic acid, and providing a signal-generating complex capable of hybridizing to the set of one or more target probes, wherein the signal-generating complex comprises a nucleic acid component capable of hybridizing to the set of one or more target probes, and a labeled probe, and hybridizing the target nucleic acid to the set of one or more target probes, and capturing the signal-generating complex in the set of one or more target probes, thereby capturing the signal-generating complex in the target nucleic acid.

[0141] In some embodiments, each set of one or more target probes includes a single probe. In other embodiments, each set of one or more target probes includes two probes. In yet another embodiment, each set of one or more target probes includes three or more probes.

[0142] In some embodiments, when each set of target probes contains a single target probe, a signal-generating complex is formed when the single target probe binds to the target nucleic acid. In other embodiments, when each set of target probes contains two target probes, a signal-generating complex is formed when both members of the target probe pair bind to the target nucleic acid.

[0143] In some specific embodiments, the ISH used herein is RNAscope®, which is described in detail, for example, by U.S. Patents 7,709,198, 8,604,182, and 8,951,726. Specifically, RNAscope® is described as being used in combination with a specially designed oligonucleotide probe and a branched DNA-like signaling complex to reliably detect RNA as small as 1 kilobase with single-molecule sensitivity under standard bright-field microscopy (Anderson et al., J. Cell. Biochem. 117(10):2201-2208 (2016), Wang et al., J. Mol. Diagn. 14(1):22-29 (2012)).

[0144] In some embodiments, each target probe comprises a target (T) section and a label (L) section, where the T section is a nucleic acid sequence complementary to the section on the target nucleic acid, and the L section is a nucleic acid sequence complementary to the section on the nucleic acid component of the signal-generating complex, where the T sections of one or more target probes are complementary to non-overlapping regions of the target nucleic acid, and the L sections of one or more target probes are complementary to non-overlapping regions of the nucleic acid component of the signal-generating complex.

[0145] In some embodiments, one set of one or more target probes is used to detect the target nucleic acid. In other embodiments, two or more sets of one or more target probes are used to detect the target nucleic acid. In some embodiments, two sets of one or more target probes are used to detect the target nucleic acid. In some embodiments, three sets of one or more target probes are used to detect the target nucleic acid. In some embodiments, four sets of one or more target probes are used to detect the target nucleic acid. In some embodiments, five sets of one or more target probes are used to detect the target nucleic acid. In some embodiments, six sets of one or more target probes are used to detect the target nucleic acid. In some embodiments, seven sets of one or more target probes are used to detect the target nucleic acid. In some embodiments, eight sets of one or more target probes are used to detect the target nucleic acid. In some embodiments, nine sets of one or more target probes are used to detect the target nucleic acid. In some embodiments, ten sets of one or more target probes are used to detect the target nucleic acid. In some embodiments, eleven or more sets of one or more target probes are used to detect the target nucleic acid. In some embodiments, target nucleic acids are detected using 16 or more sets of one or more target probes. In some embodiments, target nucleic acids are detected using 21 or more sets of one or more target probes. In some embodiments, target nucleic acids are detected using 31 or more sets of one or more target probes.

[0146] In some embodiments, the methods provided herein are for detecting multiple nucleic acid targets. In some embodiments, all of the multiple nucleic acid targets contain fewer than 100 nucleotides. In other embodiments, some of the nucleic acid targets contain fewer than 100 nucleotides, while others contain more than 100 nucleotides. In other embodiments, some of the multiple nucleic acid targets contain more than 1000 nucleotides.

[0147] As used herein, “target probe” is a polynucleotide that can hybridize to a target nucleic acid to capture or bind components of a labeled probe or signal-generating complex (SGC) to that target nucleic acid. A target probe can hybridize directly to a labeled probe or to one or more nucleic acids that sequentially hybridize to a labeled probe. For example, a target probe can hybridize to an amplifier, pre-amplifier, or pre-pre-amplifier in an SGC. That is, a target probe comprises a first polynucleotide sequence complementary to the polynucleotide sequence of the target nucleic acid, and a second polynucleotide sequence complementary to a polynucleotide sequence such as a labeled probe, amplifier, pre-amplifier, or pre-pre-amplifier. Target probes are generally single-stranded, and the complementary sequences are available for hybridization with the corresponding target nucleic acid, labeled probe, amplifier, pre-amplifier, or pre-pre-amplifier. In some embodiments, target probes are supplied as pairs.

[0148] As used herein, the term “labeled probe” refers to an object that binds directly or indirectly, or substantially indirectly, to a target molecule, thereby making the target detectable. A labeled probe (or “LP”) comprises a nucleic acid binding portion, typically a single-stranded polynucleotide or oligonucleotide, which includes one or more labels that provide a directly or indirectly detectable signal. The labels may be covalently bound to the polynucleotide, or the polynucleotide may be configured to bind to the labels. For example, a biotinylated polynucleotide can bind to a streptavidin-binding label. A labeled probe can, for example, directly hybridize to a target nucleic acid. Generally, a labeled probe can hybridize to nucleic acids that are sequentially hybridizing to the target nucleic acid, or to one or more other nucleic acids that are hybridizing to the target nucleic acid. That is, a labeled probe may include a polynucleotide sequence of the target nucleic acid, in particular a polynucleotide sequence complementary to a portion of the target nucleic acid. Alternatively, a labeled probe may include at least one polynucleotide sequence complementary to the polynucleotide sequence of an amplifier, pre-amplifier, or pre-pre-amplifier in the SGC.

[0149] In some embodiments, the SGCs provided herein include additional descriptions with respect to such amplifiers, pre-amplifiers, and / or pre-pre-amplifiers.

[0150] As used herein, “amplifier” is a molecule, typically a polynucleotide, that can hybridize to multiple labeled probes. Typically, an amplifier hybridizes to multiple identical labeled probes. An amplifier may also hybridize to a target nucleic acid, at least one target probe of a target probe pair, both target probes of a target probe pair, or nucleic acids (such as an amplifier, pre-amplifier, or pre-pre-amplifier) ​​bound to a target probe. For example, an amplifier can hybridize to at least one target probe and multiple labeled probes, or to a pre-amplifier and multiple labeled probes. An amplifier can be, for example, a linear, fork-shaped, comb-shaped, or branched nucleic acid. As described herein for all polynucleotides, an amplifier may include modified nucleotides and / or non-standard internucleotide bonds, as well as standard deoxyribonucleotides, ribonucleotides, and / or phosphodiester bonds. Suitable amplifiers are described, for example, in U.S. Patents 5,635,352, 5,124,246, 5,710,264, 5,849,481, and 7,709,198, and U.S. Publications 2008 / 0038725 and 2009 / 0081688, which are incorporated by reference, respectively.

[0151] As used herein, “pre-amplifier” refers to a molecule, typically a polynucleotide, that acts as an intermediate binding component between one or more target probes and one or more amplifiers. Typically, the pre-amplifier hybridizes simultaneously with one or more target probes and multiple amplifiers. Exemplary pre-amplifiers are described, for example, in U.S. Patents 5,635,352, 5,681,697, and 7,709,198, and U.S. Publications 2008 / 0038725, 2009 / 0081688, and 2017 / 0101672, which are invoked by reference, respectively.

[0152] As used herein, “pre-pre-amplifier” refers to a molecule, typically a polynucleotide, that acts as an intermediate binding component between one or more target probes and one or more pre-amplifiers. Typically, the pre-pre-amplifier hybridizes simultaneously with one or more target probes and multiple pre-amplifiers. An example pre-pre-amplifier is described, for example, in 2017 / 0101672, which is incorporated by reference.

[0153] Labels are typically used in ISH to detect target nucleic acids. As used herein, “label” refers to a portion that facilitates the detection of a molecule. Common labels include fluorescent labels, luminescent labels, light-scattering labels, and / or colorimetric labels. Preferred labels include enzymes, as well as fluorescent and color-emitting moieties, and radionuclides, substrates, cofactors, inhibitors, chemiluminescent moieties, magnetic particles, rare earth metals, and metal isotopes. In certain embodiments, the label is an enzyme. Exemplary enzyme labels include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, and glucose oxidase, as well as various proteases. Other labels include, but are not limited to, fluorophores and dinitrophenyl (DNP). Labels are well known to those skilled in the art, for example, as described in Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996), and U.S. Patents No. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241. Many labels are commercially available, including detectable enzyme / substrate combinations (Pierce, Rockford IL, Santa Cruz Biotechnology, Dallas TX, Life Technologies, Carlsbad CA), and can be used in the methods and assays of this disclosure. In certain embodiments of this disclosure, the enzyme can generate a detectable signal using a chromogenic or fluorescent substrate, as described herein. Exemplary labels are described herein.

[0154] Any number of enzyme-active or non-enzymatic labels can be used, as long as each is detectable. The enzyme generates a detectable signal, which can then be used to detect target nucleic acids. Particularly useful detectable signals are chromogenic or fluorescent signals. Therefore, enzymes particularly useful for use as labels include those for which a chromogenic or fluorescent substrate is available. Such chromogenic or fluorescent substrates can be readily converted by enzymatic reactions into detectable chromogenic or fluorescent products, which can then be readily detected and / or quantified using microscopy or spectroscopy. Such enzymes are well known to those skilled in the art and include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and glucose oxidase (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Other enzymes with well known chromogenic or fluorescent substrates include various peptidases, whose chromogenic or fluorescent peptide substrates can be used to detect protein cleavage reactions. The use of chromogenic and fluorescent substrates is well known in bacterial diagnosis, and examples include, but is not limited to, α-galactosidase, β-galactosidase, β-glucuronidase, 6-phospho-β-D-galactoside6-phosphogalactohydrolase, β-glucosidase, α-glucosidase, amylase, neuraminidase, esterase, and lipase. (Manafi et al., Microbiol. Rev. 55: 335-348 (1991)) Any such enzyme having a known chromogenic or fluorescent substrate can be readily adapted for use in the methods provided herein.

[0155] Various chromogenic or fluorescent substrates for generating detectable signals are well known to those skilled in the art and are commercially available. Exemplary substrates that can be used to generate detectable signals include: 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), chloronaphthol (4-CN) (4-chloro-1-naphthol), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine dihydrochloride (OPD), and 3-amino-9-ethylcarbazole (AEC) for horseradish peroxidase; 5-bromo-4-chloro-3-indolyl-1-phosphate (BCIP), nitrobluetetrazolium (NBT), and Fast Red (Fast Red Examples of fluorescent substrates include, but are not limited to, TR / AS-MX) and p-nitrophenyl phosphate (PNPP), 1-methyl-3-indolyl-β-D-galactopyranoside and 2-methoxy-4-(2-nitrovinyl)phenyl β-D-galactopyranoside for β-galactosidase, and 2-methoxy-4-(2-nitrovinyl)phenyl β-D-glucopyranoside for β-glucosidase. Examples of fluorescent substrates include, but are not limited to, 4-(trifluoromethyl)umbelliferyl phosphate for alkaline phosphatase, 4-methylumbelliferyl phosphate bis(2-amino-2-methyl-1,3-propanediol), 4-methylumbelliferyl phosphate bis(cyclohexylammonium) and 4-methylumbelliferyl phosphate for phosphatase, QuantaBlu® and Quintolet for horseradish peroxidase, and for β-galactosidase. Examples of such agents include, but are not limited to, 4-methylumbelliferyl-β-D-galactopyranoside, fluoresceindi(β-D-galactopyranoside) and naphthofluoresceindi-(β-D-galactopyranoside), 3-acetylumbelliferyl-β-D-glucopyranoside and 4-methylumbelliferyl-β-D-glucopyranoside for β-glucosidase, and 4-methylumbelliferyl-α-D-galactopyranoside for α-galactosidase.Exemplary enzymes and substrates that generate detectable signals are also described, for example, in U.S. Publication No. 2012 / 0100540. Various detectable enzyme substrates, including chromogenic substrates or fluorescent substrates, are well-known and commercially available (Pierce, Rockford IL, Santa Cruz Biotechnology, Dallas TX, Invitrogen, Carlsbad CA, 42 Life Science, Biocare). Generally, the substrate is converted to a product that forms a precipitate that deposits at the site of the target nucleic acid. Other exemplary substrates include, but are not limited to, HRP-Green (42 Life Science), Betazoid DAB, Cardassian DAB, Romulin AEC, Bajoran Purple, Vina Green, Deep Space Black (trademark), Warp Red (trademark), Vulcan Fast Red and Ferangi Blue manufactured by Biocare (Concord CA; biocare.net / products / detection / chromogens).

[0156] Exemplary rare earth metals and metal isotopes suitable as detectable labels include 141 Pr, 142 Nd, 143 Nd, 144 Nd, 145 Nd, 146 Nd, 147 Sm, 148 Nd, 149 Sm, 150 Nd, 151 Eu, 152 Sm, 153 Eu, 154 Sm, 155 Gd, 156 Gd, 158 Gd, 159 Tb, 160 Gd, 161 Dy, 162 Dy, 163 Dy, 164 Dy, 165 Ho, 166 Er, 167 Er, 168 [[ID=5169 Tm, 170 Er, 171 Yb, 172 Yb, 173 Yb, 174 Yb, 175 Lu, and 176 Examples include, but are not limited to, lanthanide(III) isotopes such as Yb. Metallic isotopes can be detected using, for example, time-of-flight mass spectrometry (TOF-MS) (e.g., Fluidigm's Helios and Hyperion systems, fluidigm.com / systems; South San Francisco, CA).

[0157] Biotin-avidin (or biotin-streptavidin) is a well-known signal amplification system based on the high affinity between the two molecules and the fact that one avidin / streptavidin molecule can bind to four biotin molecules. Antibodies are widely used for signal amplification in immunohistochemistry and ISH. Tyramide signal amplification (TSA) is based on the deposition of numerous haptenized tyramide molecules by peroxidase activity. Tyramine is a phenolic compound. Immobilized horseradish peroxidase (HRP), in the presence of a small amount of hydrogen peroxide, converts the labeled substrate into a highly reactive, short-lived intermediate. Subsequently, the activated substrate molecule reacts very rapidly with an electron-rich protein moiety (such as tyrosine) at or near the peroxidase binding site, and covalently binds to that moiety. This method allows for the in situ introduction of numerous hapten molecules conjugated to tyramide at the hybridization site. The deposited tyramide-hapten molecules can then be visualized directly or indirectly. Such detection systems are described in more detail, for example, in U.S. Publication No. 2012 / 0100540.

[0158] In the embodiments described herein, enzymes can be used to generate a detectable signal using a suitable chromogenic or fluorescent substrate. Alternatively, it is understood that a labeled probe may have a detectable label directly bound to the nucleic acid portion of the labeled probe. Exemplary detectable labels are well known to those skilled in the art and include, but are not limited to, chromogenic or fluorescent labels (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Examples of fluorophores useful as labels include rhodamine derivatives, e.g., tetramethylrhodamine, rhodamine B, rhodamine 6G, sulforhodamine B, Texas Red (sulforhodamine 101), rhodamine 110, and their derivatives, e.g., tetramethylrhodamine-5-(or 6), lysaminerhodamine B, etc.; 7-nitrobenz-2-oxa-1,3-diazole (NBD); fluorescein and its derivatives; naphthalene, e.g., dansyl (5-dimethylaminonaphthalene-1-sulfonyl); coumarin derivatives, e.g., 7-amino-4-methylcoumarin-3-acetic acid (AMCA), 7-diethylamino-3-[(4'-(iodoacetyl)amino)phenyl]-4-methylcoumarin (DCIA), and Alexa fluorescent dyes (Molecular Probes) etc; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY®) and its derivatives (Molecular Probes; Eugene, OR); pyrene and sulfonated pyrene, e.g., Cascade Blue® and its derivatives, e.g., 8-methoxypyrene-1,3,6-trisulfonic acid; pyridyloxazole derivatives and dapoxyl derivatives (Molecular Probes); Lucifer Yellow (3,6-disulfonate-4-amino-naphthalimide) and its derivatives; CyDye® fluorescent dye (Amersham / GE Healthcare Life Sciences;Piscataway NJ), ATTO 390, DyLight 395XL, ATTO 425, ATTO 465, ATTO 488, ATTO 490LS, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 643, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO Examples of chromophores include, but are not limited to, 680, ATTO 700, ATTO 725, ATTO 740, and Cyan 500 NHS-Ester (ATTO-TECH, Siegen, Germany). Exemplary chromophores include, but are not limited to, phenolphthalein, malachite green, aromatic nitro compounds (such as nitrophenyl), diazo dyes, and dabucil (4-dimethylaminoazobenzene-4'-sulfonyl).

[0159] As disclosed herein, the methods provided herein can be used for the simultaneous detection of multiple target nucleic acids. When fluorophores are used as labels, the fluorophores used for the detection of multiple target nucleic acids are selected so that each fluorophore is identifiable in the case of simultaneous detection of target nucleic acids and so that the fluorophores can be detected simultaneously with a fluorescence microscope. Such fluorophores are selected so that the emission spectrum is separated so that separate labels of target nucleic acids can be detected simultaneously. Methods for selecting identifiable fluorophores suitable for use in the methods disclosed herein are well known in the art (see, for example, Johnson and Spence, “Molecular Probes Handbook, a Guide to Fluorescent Probes and Labeling Technologies,” 11th ed., Life Technologies (2010)).

[0160] Well-known methods such as microscopy, cytometry (e.g., mass cytometry, time-of-flight cytometry (CyTOF), flow cytometry), or spectroscopy can be used to visualize detectable chromogenic, fluorescent, or metallic signals associated with each target nucleic acid. Generally, to allow the use of a single instrument for detecting nucleic acid targets in the same sample, if different labels are used in the same assay, either a chromogenic substrate or a fluorescent substrate, or a chromogenic label or a fluorescent label, or a rare-earth metal isotope is used, depending on the specific assay.

[0161] As disclosed herein, labels can be designed to be optionally cleavable. As used herein, a cleavable label refers to a label that is conjugated or conjugated to a labeling probe so that the label can be removed, for example, in a second or subsequent round of labeling and detecting a target nucleic acid. Generally, the label is conjugated to the labeling probe by a cleavable chemical linker. Methods for conjugating a label to a labeling probe so that the label is cleavable are well known to those skilled in the art (see, e.g., Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996), Daniel et al., BioTechniques 24(3):484-489 (1998)). One specific system for labeling oligonucleotides is the FastTag® system (Daniel et al., 1998, Vector Laboratories, Burlingame CA). Various cleavable moieties can be included in the linker to allow the label to be cleaved from the labeled probe. Such cleavable moieties include groups that can be cleaved chemically, photochemically, or enzymatically. A cleavable chemical linker may include cleavable chemical moieties such as disulfides that can be cleaved by reduction, glycols or diols that can be cleaved by periodates, diazo bonds that can be cleaved by dithionites, esters that can be cleaved by hydroxylamines, and sulfones that can be cleaved by bases (see Hermanson, 1996 above). One particularly useful cleavable linker is one that includes a disulfide bond that can be cleaved by reduction. In another embodiment, the linker may include sites for enzymatic cleavage. For example, the linker may include protein cleavage sites. Generally, such cleavage sites are for sequence-specific proteases.Examples of such proteases include, but are not limited to, human rhinovirus 3C protease (cleavage site: LEVLFQ / GP), enterokinase (cleavage site: DDDDK / ), factor Xa (cleavage site: IEGR / ), tobacco etch virus protease (cleavage site: ENLYFQ / G), and thrombin (cleavage site: LVPR / GS) (see, e.g., Oxford Genetics, Oxford, UK). Another cleavable region may be uracil-DNA (uracil-containing DNA) that can be cleaved by uracil-DNA glycosylase (UNG) (see, e.g., Sidorenko et al., FEBS Lett. 582(3):410-404 (2008)).

[0162] A cleavable label can be removed by applying a chemical agent or light to cleave the label and dissociate it from the label probe. As discussed above, useful cleaving agents for chemical cleavage include, but are not limited to, reducing agents, periodic acid, dithionites, hydroxylamines, and bases (see Hermanson, 1996 above). One useful method for cleaving a linker containing a disulfide bond is to utilize tris(2-carboxyethyl)phosphine (TCEP) (see Moffitt et al., Proc. Natl. Acad. Sci. USA 113:11046-11051 (2016)). In one embodiment, TCEP is used as the agent to cleave the label from the label probe.

[0163] In some embodiments, the methods provided herein involve the use of a labeled primary antibody, thereby eliminating the need to perform other IHC steps. In certain embodiments, the primary antibody is labeled with a chromogenic label. In certain embodiments, the primary antibody is labeled with a fluorescent label. In certain embodiments, the primary antibody is labeled with a polynucleotide. In certain embodiments, the primary antibody is labeled with a polynucleotide. In certain embodiments, the primary antibody is labeled by the NHS (succinimidyl) ester method. In certain embodiments, the primary antibody is labeled by the isothiocyanate method. In certain embodiments, the primary antibody is labeled by the carbodiimide method. In certain embodiments, the primary antibody is labeled by the two-tag method (catalyst and substrate). In certain embodiments, the primary antibody is labeled by the periodic acid method.

[0164] For use with fluorescence-based detection, crosslinking after primary antibody can be employed in combination with the BaseScope® signal amplification system (see Baker et al., Nature Communications 8:1998(2017)) or in combination with other nucleic acid detection methods using similar protocols. In some embodiments, the methods provided herein are for preparing a biological sample for the simultaneous detection of target RNA and target protein, and include (i) incubation of the biological sample with a primary antibody, (ii) treatment of the biological sample with a crosslinking agent after (i), and (iii) detection of the target RNA by a hybridization-based method after (ii). In one embodiment, the method includes detecting the target RNA by ISH. In one embodiment, the method includes detecting the target RNA by molecular beacon (see Tyagi et al., Nat Biotechnol. 4(3):303-308(1996)). In one embodiment, the method includes detecting the target RNA by a forced intercalation (FIT) probe (see Kohler et al., Chembiochem. 6(1):69-77 (2005)). In some embodiments, the method provided herein is for preparing a biological sample for the simultaneous detection of a target RNA and a target protein, and includes (i) incubating the biological sample with a primary antibody, (ii) treating the biological sample with a crosslinking agent after (i), and (iii) detecting the target RNA by an aptamer-based method after (ii). In one embodiment, the method includes detecting the target RNA by the aptamer "Spinach" (see Paige et al., Science, 333(6042):642-646 (2011)).In some embodiments, the methods provided herein are for preparing a biological sample for the simultaneous detection of target RNA and target protein, and include (i) incubating the biological sample with a primary antibody, (ii) treating the biological sample with a crosslinking agent after (i), and (iii) detecting the target RNA with a particle-associated hybridization-based probe after (ii). In one embodiment, the method includes detecting the target RNA with a gold nanoparticle quantum dot probe. In some embodiments, the methods provided herein are for preparing a biological sample for the simultaneous detection of target RNA and target protein, and include (i) incubating the biological sample with a primary antibody, (ii) treating the biological sample with a crosslinking agent after (i), and (iii) detecting the target RNA by directly incorporating a visualizeable portion into the target RNA (see Jao et al., Proc Natl Acad Sci. 105(41):15779-15784 (2008)). In some embodiments, the methods provided herein are for preparing a biological sample for the simultaneous detection of a target RNA and a target protein, and include (i) incubating the biological sample with a primary antibody, (ii) treating the biological sample with a crosslinking agent after (i), and (iii) detecting the target RNA with an RNA-binding protein. In one embodiment, the method includes detecting the target RNA by a fluorescent protein fusion version of bacteriophage MS2 coat protein (MCP) (see Park et al., Science. 343(6169):422-424 (2014)). In one embodiment, the method includes detecting the target RNA by a Pumilio-based method (see Kellermann et al., Chembiochem. 14(2):200-204 (2013)).

[0165] Since similar proteases and hybridization conditions that may impair antigen-antibody binding can be used for DNA detection, the crosslinking methods described herein also enable co-detection of protein-DNA. In some embodiments, the methods provided herein are for preparing a biological sample for the simultaneous detection of target DNA and target protein, and include (i) incubation of the biological sample with a primary antibody, (ii) treatment of the biological sample with a crosslinking agent after (i), and (iii) detection of the target DNA after (ii) by a hybridization-based method. In one embodiment, the method includes detecting the target DNA by chromogenic ISH. In one embodiment, the method includes detecting the target DNA by fluorescent ISH.

[0166] The methods provided herein are useful research tools, as well as diagnostic tools. In some embodiments, the methods provided herein are used to map the spatial configuration in composite tissues. In some specific embodiments, the methods provided herein are used to identify cell types and novel cell types. In some specific embodiments, the methods provided herein are used to identify cellular states. In other specific embodiments, the methods provided herein are used to identify cell types and novel cell types in the tumor microenvironment. In some specific embodiments, the methods provided herein are used to identify cellular states in the tumor microenvironment.

[0167] In some embodiments, the methods provided herein are used to detect denatured gene expression in diseased cells and tissues. In some specific embodiments, the methods provided herein are used to locate denatured gene expression in specific cell types and to understand intratumoral heterogeneity. In some specific embodiments, the methods provided herein are used to study the interactions of tumor immune cells. In some embodiments, the methods provided herein are used to detect biomarkers for cancer diagnosis and prognosis. In some embodiments, the methods provided herein are used to detect therapeutic targets for cancer treatment. In some embodiments, the methods provided herein are used to facilitate the validation of novel antibodies.

[0168] 6.4 Kits for simultaneous detection of target nucleic acids and target proteins In another embodiment, this specification provides kits for carrying out the various methods described in sections 6.2 and 6.3 above.

[0169] In another embodiment, this specification provides a kit for the simultaneous detection of a target nucleic acid and a target protein in a biological sample, comprising (i) a crosslinking agent and (ii) instructions for use indicating that the crosslinking agent is used after the biological sample has been incubated with a primary antibody for detecting the target protein. In some embodiments, the kit further comprises a protease. In some embodiments, the kit further comprises an agent for detecting the target nucleic acid and / or an agent for detecting the target protein.

[0170] In another embodiment, this specification provides a kit for simultaneously detecting a target nucleic acid and a target protein in a biological sample, comprising (i) a crosslinking agent and (ii) a protease. In some embodiments, the kit further includes instructions indicating that the crosslinking agent is used before the protease and that the crosslinking agent is used after the primary antibody for detecting the target protein. In some embodiments, the kit further includes an agent for detecting the target nucleic acid and / or an agent for detecting the target protein.

[0171] In some embodiments, the crosslinking agent in the kit is a fixative. In certain embodiments, the crosslinking agent in the kit is formaldehyde. In certain embodiments, the crosslinking agent in the kit is glutaraldehyde. In certain embodiments, the crosslinking agent in the kit is acrolein. In certain embodiments, the crosslinking agent in the kit is osmium tetroxide. In certain embodiments, the crosslinking agent in the kit is a type of permanganate fixative. In one embodiment, the crosslinking agent in the kit is potassium permanganate. In certain embodiments, the crosslinking agent in the kit is a type of dichromate fixative. In one embodiment, the crosslinking agent in the kit is potassium dichromate. In certain embodiments, the crosslinking agent in the kit is chromic acid.

[0172] In some embodiments, the kits provided herein include a crosslinking agent, which is a mixture of crosslinkable fixatives. In some embodiments, the crosslinking agent is a mixed solution of two or more fixatives selected from the list of formaldehyde, glutaraldehyde, acrolein, osmium tetroxide, permanganate fixatives, dichromate fixatives, and chromic acid. In one embodiment, the crosslinking agent is Bouin fixative, which is a solution of picric acid, formaldehyde, and acetic acid. In one embodiment, the crosslinking agent is a mixture of formaldehyde and glutaraldehyde. In one embodiment, the crosslinking agent is FAA, which is a solution of ethanol, acetic acid, and formaldehyde. In one embodiment, the crosslinking agent is periodate-lysine-paraformaldehyde (PLP), which is a solution of paraformaldehyde, L-lysine, and INaO4. In one embodiment, the crosslinking agent is phosphate-buffered formalin (PBF). In one embodiment, the crosslinking agent is formal calcium, which is a solution of formaldehyde and calcium chloride. In one embodiment, the crosslinking agent is formal saline solution, which is a solution of formaldehyde and sodium chloride. In one embodiment, the crosslinking agent is zinc formalin, which is a solution of formaldehyde and zinc sulfate. In one embodiment, the crosslinking agent is Helly fixative, which is a solution of formaldehyde, potassium dichromate, sodium sulfate, and mercury chloride. In one embodiment, the crosslinking agent is Hollande fixative, which is a solution of formaldehyde, copper acetate, picric acid, and acetic acid. In one embodiment, the crosslinking agent is Gendre solution, which is a solution of formaldehyde, ethanol, picric acid, and glacial acetic acid. In one embodiment, the crosslinking agent is alcohol formalin, which is a solution of formaldehyde, ethanol, and calcium acetate. In one embodiment, the crosslinking agent is formal acetate alcohol, which is a solution of formaldehyde, glacial acetic acid, and ethanol. In one embodiment, the crosslinking agent is a mixture of fixatives, at least one of which is formaldehyde or glutaraldehyde. In one embodiment, the crosslinking agent is a fixative that is not used simultaneously but is used separately or sequentially, and at least one of the fixatives is formaldehyde or glutaraldehyde.

[0173] In some embodiments, the kits provided herein include two or more crosslinking agents, which are not applied simultaneously but separately or sequentially, and are selected from the list of formaldehyde, glutaraldehyde, acrolein, osmium tetroxide, permanganate fixatives, dichromate fixatives, and chromic acid.

[0174] In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 0°C to about 100°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 1°C to about 90°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 2°C to about 80°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 3°C ​​to about 70°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 4°C to about 60°C.

[0175] In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 1°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 2°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 3°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 4°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 5°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 6°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 7°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 8°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 9°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 10°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 11°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 12°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 13°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 14°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 15°C.In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 16°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 17°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 18°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 19°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 20°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 21°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 22°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 23°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of approximately 24°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of approximately 25°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of approximately 26°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of approximately 27°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of approximately 28°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of approximately 29°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of approximately 30°C.In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 35°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 40°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 45°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 50°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 55°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 60°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 65°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 70°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 75°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 80°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 85°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 90°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 95°C. In some embodiments, the kit provided herein includes instructions indicating that the biological sample is treated with a crosslinking agent at a temperature of about 100°C.

[0176] In some embodiments, the kit further includes tools for obtaining biological samples from the subject. In certain embodiments, the biological sample is a tissue sample or derived from a tissue sample. In certain embodiments, the biological sample is a blood sample or derived from a blood sample. In certain embodiments, the biological sample is a cytological sample or derived from a cytological sample.

[0177] In certain embodiments, the kits provided herein include agents for performing RNAscope®, as described in detail, for example, by U.S. Patents 7,709,198, 8,604,182, and 8,951,726. In some embodiments, the kit includes at least one set of one or more target probes capable of hybridizing to a target nucleic acid, and a signal-generating complex capable of hybridizing to the set of one or more target probes, the signal-generating complex comprising a labeled probe and a nucleic acid component capable of hybridizing to the set of one or more target probes.

[0178] In some embodiments, a target probe(s) comprises a target (T) section and a label (L) section, where the T section is a nucleic acid sequence complementary to a section on the target nucleic acid, and the L section is a nucleic acid sequence complementary to a section on a nucleic acid component of the signal-generating complex, where the T sections of one or more target probes(s) are complementary to non-overlapping regions of the target nucleic acid, and the L sections of one or more target probes(s) are complementary to non-overlapping regions of the nucleic acid components of the signal-generating complex.

[0179] In some embodiments, the kit further comprises a signal-generating complex as described in Section 6.3 above, which may include a labeled probe, an amplifier, a pre-amplifier, and / or a pre-pre-amplifier.

[0180] In some embodiments, the kit further includes fixatives and agents for treating the sample in preparation for hybridization, as well as other agents or materials for performing ISH, such as agents for washing the sample.

[0181] In some embodiments, the kit further includes other agents or materials for performing IHC, such as blocking buffer, secondary antibody, IHC label, and agent for washing the sample.

[0182] This kit may further include “packaging material,” which means a physical structure that contains the components of this kit. The packaging material can maintain the sterility of the components and can be made from materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0183] The kits provided herein may include labels or inserts. These labels or inserts may include information about the conditions, disorders, diseases, or symptoms for which the kit components may be used. The labels or inserts may also include instructions for use by a clinician or subject in a method, treatment protocol, or treatment regimen for the use of one or more of the kit components.

[0184] In some embodiments, the kits provided herein are used to map the spatial configuration in composite tissue. In some specific embodiments, the kits provided herein are used to identify cell types and novel cell types. In some specific embodiments, the kits provided herein are used to identify cellular states. In other specific embodiments, the kits provided herein are used to identify cell types and novel cell types in the tumor microenvironment. In some specific embodiments, the kits provided herein are used to identify cellular states in the tumor microenvironment.

[0185] In some embodiments, the kits provided herein are used to detect denatured gene expression in diseased cells and tissues. In some specific embodiments, the kits provided herein are used to locate denatured gene expression in specific cell types and to understand intratumoral heterogeneity. In some specific embodiments, the kits provided herein are used to study the interactions of tumor immune cells. In some embodiments, the kits provided herein are used to detect biomarkers for cancer diagnosis and prognosis. In some embodiments, the kits provided herein are used to detect therapeutic targets for cancer treatment. In some embodiments, the kits provided herein are used to facilitate the validation of novel antibodies.

[0186] 7. Examples The following describes various methods and materials used in the research and is presented to provide a complete disclosure and explanation of how to prepare and use this disclosure to those skilled in the art, and is not intended to limit the scope of what the inventors consider to be their disclosure, nor is it intended to indicate that the experiments described below have been performed and are not all possible. Illustrative descriptions written in the present tense should be understood not as necessarily having been performed, but rather as examples of how these descriptions can be performed to produce data relevant to the teachings of this disclosure. While efforts have been made to ensure accuracy of the numerical values ​​used (e.g., quantities, percentages, etc.), some experimental errors and variability should be taken into consideration.

[0187] 7.1 Integrated ISH / IHC Co-detection Workflow Figures 1A and 1B illustrate the steps of the sequential ISH-IHC workflow and the integrated dual ISH-IHC workflow. The original sequential dual ISH-IHC workflow begins with standard RNAscope® pretreatment and completes ISH staining before initiating IHC staining (see Figure 1A). In contrast, the integrated ISH-IHC co-detection workflow begins only with pretreatment up to target antigen retrieval. Next, the IHC primary antibody is bound to and crosslinked with the antigen in the tissue / cell, followed by protease treatment and RNAscope® staining. Once RNAscope® staining is complete, IHC detection is resumed by exposure to the secondary antibody and staining (see Figure 1B). Theoretically, using a primary antibody directly labeled for IHC could eliminate the need for exposure to the secondary antibody and potentially shorten this integrated workflow.

[0188] 7.2 Preservation or improvement of IHC signal after exposure to hybridization buffer or protease treatment by crosslinking after primary antibody treatment. The CD20 IHC signal was protected from stress by formamide-based reagents in hybridization buffer by crosslinking. Figures 2A–2C show FFPE human tonsil tissue sections stained with IHC using the Leica Bond Polymer Refine Detection Kit. As a reference, IHC for CD20 detection without any interference from ISH showed a clear signal (see Figure 2A). After incubation with CD20 antibody, the sections were exposed to hybridization buffer at room temperature for 30 minutes, followed by remaining IHC staining. Exposure to hybridization buffer significantly reduced the CD20 IHC signal (see Figure 2B). Crosslinking after primary antibody was achieved by incubating the tissue with commercial 10% NBF at room temperature for 30 minutes, which improved the resistance of IHC to the effects of hybridization buffer (see Figure 2C).

[0189] Similarly, crosslinking also improved the resistance of the CD20 IHC signal to protease treatment. Using the Leica Bond Polymer Refine Detection kit, human tonsil sections were stained with IHC to detect the CD20 protein, and a clear signal was obtained by staining (see Figure 3A). After incubation with the primary antibody, the slides were exposed to protease treatment at 40°C for 15 minutes, resulting in a significant reduction of the IHC signal (see Figure 3B). Crosslinking the tissue with 10% NBF at room temperature for 30 minutes after incubation with the primary antibody and before protease treatment improved the effect of protease on the IHC signal (see Figure 3C).

[0190] For several other antibodies studied, crosslinking after primary antibody incubation not only protected but also enhanced the IHC signal. One example was the CD8 antibody. Human tonsil sections were stained with IHC using the Leica Bond Polymer Refine Detection kit to detect CD8 (see Figures 4A and 4D). After incubation of the tissue with the CD8 antibody, exposure to hybridization buffer at room temperature for 30 minutes (see Figure 4B) or protease treatment at 40°C for 15 minutes (see Figure 4E) resulted in a significant reduction of the IHC signal. Crosslinking the tissue with 10% NBF at room temperature for 30 minutes after primary antibody incubation and before either hybridization buffer incubation (see Figure 4C) or protease treatment (see Figure 4F) improved the CD8 IHC signal compared to the standard IHC protocol (see Figures 4A and 4D, respectively).

[0191] 7.3 Strong ISH and IHC signals successfully obtained by the ISH / IHC integrated co-detection workflow As a reference for IHC or ISH alone, IHC staining for CD8 was performed on FFPE human head and neck cancer sections using the Leica Bond Polymer Refine Detection kit, and the detection of the IHC signal was visualized (see Figure 5A). Separately, ISH staining for human peptidyl prolyl isomerase B (Hs-PPIB) was performed alone using the RNAscope® 2.5 LS Red kit, and the ISH signal was also detected (see Figure 5B). However, when using a sequential ISH-IHC workflow (see Figure 1A) in which ISH detection of Hs-PPIB with RNAscope® 2.5 LS Red was performed on the sections, followed immediately by IHC staining for CD8 using the Leica Bond Polymer Refine Detection kit, the IHC signal was significantly reduced (see Figure 5C). In contrast, when using the integrated co-detection workflow (see Figure 1B), the tissue was crosslinked after exposure to the primary antibody, as described above. Next, the tissue was stained for Hs-PPIB using the RNAscope® 2.5 LS Red assay, followed by the remaining IHC staining steps. Therefore, the integrated ISH / IHC workflow helped preserve antigen-antibody binding and, in the case of CD8, improved the IHC signal (see Figure 5D).

[0192] There are more than one differences between sequential ISH / IHC and integrated co-detection workflows. To further identify which(s) are responsible for the better ISH and IHC signals when using the ISH / IHC integrated co-detection workflow, a different experimental setup was performed. FFPE human tonsil tissue sections were stained for CD20 using the Leica Bond Polymer Refine Detection kit, followed by staining with Green chromogen to detect the CD20 protein (see Figure 6A), or ISH staining for Hs-PPIB using the RNAscope® 2.5 LS Red kit to detect RNA (see Figure 6B). Alternatively, before IHC staining, sections were pre-treated with RNAscope®, including 15 minutes of incubation with protease at 40°C, resulting in a reduction of the IHC signal (see Figure 6C). When using the ISH / IHC integrated co-detection workflow (see Figure 1B), the tissue was crosslinked as described above after exposure to the primary antibody. ISH staining for Hs-PPIB was performed using the RNAscope® 2.5 LS Red assay, followed by the remaining IHC staining steps (secondary antibody, chromogenic detection, and counterstaining). Crosslinking in the integrated ISH / IHC workflow helped preserve antigen-antibody binding and improved the IHC signal (see Figure 6E). In contrast, performing an integrated ISH / IHC codetection workflow without additional crosslinking resulted in a uniform loss of the CD20 IHC signal, highlighting the importance of crosslinking after primary antibody (see Figure 6D).

[0193] 7.4 Temperature and processing duration parameters for crosslinking in an integrated ISH / IHC codetection workflow Considering that the crosslinking process is a key step in the ISH / IHC integrated co-detection workflow, more experiments were conducted to test the feasibility of different temperature and duration combinations. First, crosslinking at room temperature was shown to be effective for 15–60 minutes. FFPE human gastric cancer tissue was stained with ISH for Hs-PPIB detection and immediately followed by IHC detection of CD8 using RNAscope® 2.5 LS Red and the Leica Bond Polymer Refine Detection kit, respectively. Using a sequential ISH-IHC co-detection workflow, ISH staining of Hs-PPIB was successful, but the detected CD8 IHC signal may have been minimal (see Figure 7A). Using the ISH / IHC integrated co-detection workflow, crosslinking at room temperature for 15 minutes (see Figure 7B), 30 minutes (see Figure 7C), and 60 minutes (see Figure 7D) resulted in successful IHC detection of CD8.

[0194] Secondly, crosslinking at heated temperatures was also shown to be effective for 15–60 minutes. FFPE human gastric cancer tissue was stained with ISH for Hs-PPIB detection as described above, followed immediately by CD8 IHC detection. Sequential ISH-IHC co-detection resulted in low CD8 IHC detection (see Figure 8A). In contrast, crosslinking at 40°C for 15 minutes (see Figure 8B), 30 minutes (see Figure 8C), or 60 minutes (see Figure 8D) within the ISH / IHC integrated co-detection workflow improved CD8 IHC detection. Crosslinking at 60°C for 15 minutes (see Figure 8E), 30 minutes (see Figure 8F), or 60 minutes (see Figure 8G) also preserved the IHC signal compared to sequential ISH-IHC co-detection, but to a lesser extent than at lower incubation temperatures (e.g., see Figures 8B, 8C, and 8D).

[0195] Thirdly, crosslinking at 4°C was also shown to be effective when performed for 2 hours or overnight. FFPE human gastric cancer tissue was stained with ISH for Hs-PPIB detection as described above, followed immediately by IHC detection of CD8. As already observed, sequential ISH-IHC co-detection negatively affected the IHC signal of CD8 (see Figure 9A). The integrated co-detection workflow was modified to allow crosslinking incubation below room temperature by performing manual tissue pretreatment, antibody incubation, and crosslinking. All subsequent steps, including RNAscope® ISH staining and the remaining IHC staining, were automated on Leica Bond Rx using RNAscope® 2.5 LS Red and Leica Bond Polymer Refine Detection kits, respectively. Compared to sequential ISH-IHC co-detection (see Figure 9A) and 30 minutes of crosslinking at room temperature (see Figure 9B), 2 hours of crosslinking at 4°C (see Figure 9C) and overnight crosslinking at 4°C (see Figure 9D) enabled successful IHC detection of CD8.

[0196] 7.5 Image Processing Embodiments of this disclosure also include methods 100 for enhancing target detection. In some embodiments, method 100 includes an image processing method. Method 100 is shown as a flowchart of the process in Figure 10B, while Figure 10A shows a plurality of images and corresponding factors used in method 100 to modify the images. In the illustrated embodiments, method 100 is carried out at least in part using a computer having corresponding instructions stored on a storage device (i.e., a non-temporary computer-readable medium). The final images from method 100, and in some embodiments, intermediate images, are stored in the storage device. In some embodiments, the storage device is accessible via a network. In some embodiments, user input or instructions are receivable or accessible via the network.

[0197] Method 100 includes imaging a sample having a target signal to create a probe image (step 104) and imaging a sample without a target signal to create a background image (i.e., a “blank image”) (step 108). In some embodiments, imaging utilizes a fluorescence microscope connected to a computer via a network. In some embodiments, the target signal is obtained by performing a fluorescent in situ hybridization assay and / or immunofluorescence assay on the sample. In some embodiments, the background image without a target signal is obtained by removing the target signal from the sample (i.e., by cleavage). In other embodiments, the background image without a target signal is obtained before the assay is performed. In other words, in some embodiments, step 104 occurs before step 108, and in other embodiments, step 104 occurs after step 108. In some embodiments, the target signal includes a fluorescent label bound to a target nucleic acid. In other embodiments, the target signal includes a fluorescent label bound to a target peptide or target polypeptide.

[0198] Continuing to refer to Figure 10B, Method 100 includes step 112 of registering probe and background images. Possible differences in background fluorescence between the probe and background images result in spatial pattern shifts that occur due to the movement of the entire sample between different rounds of image acquisition. To eliminate such differences, image registration techniques (e.g., phase correlation) are used. Robust image registration (e.g., step 112) compensates for any overall sample movement (i.e., translation and rotation) by utilizing the detection and matching of image features.

[0199] Method 100 further includes modifying a background image based on at least one image metric to create an adjusted background image (e.g., a transformed, intensity-adjusted blank image) (step 136). As further described herein, the at least one image metric is a ratio factor (steps 116, 120, 124), a doubling rate (step 128), a local maximum transformation (step 132), and any other suitable metric. In some embodiments, Method 100 includes a single image metric. In other embodiments, Method 100 includes a combination of image metrics.

[0200] Continuing to refer to Figure 10B, Method 100 further includes removing the adjusted background image from the probe image to create a final image containing the enhanced target signal (Step 140). In other words, a modified (i.e., transformed, adjusted, scaled, etc.) blank image is used in place of the original blank image in the removal step (Step 140). In some embodiments, the enhanced target signal includes contrast enhancement. In some embodiments, Method 100 includes displaying the final image on a display (e.g., a computer display) (Step 144). The final image may be stored in a storage device and made accessible to the user, for example, over a network. Thus, Method 100 provides improved signal detection in the presence of a background with tissue autofluorescence.

[0201] In some embodiments, the image metric is a ratio factor that is responsible for the background intensity difference between a blank image and a probe image. The intensity difference may arise from different image acquisition settings or from photobleaching during fluorophore excitation. To compensate for the background intensity difference, Method 100 includes steps 116, 120, and 124 for determining a ratio factor that compares the overall background intensity of the probe image versus the blank image. First, the pixel positions of the probe are estimated (step 116). The probe positions in the probe image are estimated using, for example, the White Top Hat algorithm (Gonzalez & Woods, 2008, Digital Image Processing), a bandpass filter (Shenoi, 2006, Introduction to Digital Signal Processing and Filter Design), or any combination of preferred methods. After determining the estimated positions of the target signal in the probe image (step 116), pixels at the estimated probe positions are removed from the probe image and the blank image (step 120), resulting in an image of background pixels only (i.e., a background-only image). In other words, step 120 includes removing the estimated position from the probe image to create a first background-only image, and removing the estimated position from the blank image (background image) to create a second background-only image.

[0202] Following the removal of the estimated probe position from both images (step 120), Method 100 includes step 124 of determining a ratio factor. In other words, statistical metrics for the probe-removed blank image and the probe-removed probe image are evaluated and incorporated into the ratio factor. As further described herein, in some embodiments, the ratio factor is used to modify the background image to create a tuned background image (step 136). In other words, modifying the background image to create a tuned background image may, in some embodiments, include scaling the background image by the ratio factor.

[0203] In some embodiments, at least one image metric is a ratio factor between a first background-only image and a second background-only image. For example, in some embodiments, the ratio factor is a first intensity relative to a second intensity, where the first intensity is determined from the first background-only image and the second intensity is determined from the second background-only image. In some embodiments, the first and second intensities used in the ratio factor are statistical metrics for any portion (including the whole) of the intensity values ​​in the image, such as a statistical mean, median, or a combination of both.

[0204] In some embodiments, the first intensity is the average of multiple pixel intensity values ​​in a first background-only image, and the second intensity is the average of multiple pixel intensity values ​​in a second background-only image. In some embodiments, the average is the average of all pixel intensity values ​​in the image. In other embodiments, the first intensity is the median of multiple pixel intensity values ​​in a first background-only image, and the second intensity is the median of multiple pixel intensity values ​​in a second background-only image. In some embodiments, the median is the median of all pixel intensity values ​​in the image. In yet another embodiment, the first intensity is the average of approximately 80% of the median (i.e., excluding the approximate top 10% and approximate bottom 10%) of all pixel intensity values ​​in the first background-only image, and the second intensity is the average of approximately 80% of the median of all pixel intensity values ​​in the second background-only image.

[0205] In some embodiments, the image metric is the doubling rate, which is the cause of any local intensity differences that may occur between a blank image and a probe image. In particular, Method 100 in the illustrated embodiment includes a step 128 for determining the doubling rate. In some embodiments, the doubling rate is in the range of about 1.0 to about 1.2. In other embodiments, the doubling rate is in the range of about 1.0 to about 1.1. As further described herein, in some embodiments, the doubling rate is used to modify the background image to create a tuned background image (step 136). In other words, modifying the background image to create a tuned background image may, in some embodiments, include scaling the background image by the doubling rate.

[0206] In some embodiments, the image metrics are local maximum transformations. In particular, method 100 in the illustrated embodiment includes a step 132 of transforming a blank image using a local maximum transformation. Even after the overall image has been registered, local background pattern shifts may remain due to, for example, image acquisition at different focal planes, insufficient adhesion of the sample to the support material (e.g., a glass slide), and partial movement of the sample between imaging sessions. To solve this problem, local shifts are corrected by transformation. In the illustrated embodiment, for each pixel of the blank image ("pixel of interest"), a neighborhood of a predetermined radius surrounding the pixel of interest is searched. The search process finds the pixel with the highest intensity, and this highest intensity is assigned to that pixel of interest. This search procedure is performed for each pixel of interest, and the neighborhood of each pixel of interest in the original blank image is searched to form a transformed blank image. As will be described in more detail herein, the transformed blank image can be used in place of the original blank image in a later removal step (i.e., step 140). In some embodiments, the predetermined radius ("match distance") is adjustable.

[0207] In some embodiments, the predetermined radius used for local maximum transformation is in the range of approximately 0 to approximately 5 pixels. In other words, local maximum transformation includes a search radius in the range of approximately 0 to 5 pixels. For example, when there is no significant local background pattern shift, a predetermined radius of 0 pixels is used. In some embodiments, the search area is simplified to reduce computation time by using eight lines placed at equal intervals at an angle (i.e., 45-degree intervals), each having a single-pixel width, and extending radially from the pixel of interest.

[0208] In some embodiments, the image metric is a block matching transformation. In particular, Method 100, in some embodiments, includes the step of transforming a blank image using a block matching transformation. In some embodiments, the block matching transformation is used instead of a local maximum transformation to solve the problem of local misalignment. In some embodiments, a block ("block of interest") is used with a predetermined block size (e.g., a 3x3 pixel block). Each block in the blank image is compared to a block of the same size in the probe image at a nearby location (i.e., within a predetermined block search size). The search determines the neighboring block that is most similar to the block of interest. Using a similarity metric that measures the similarity of blocks, the neighboring block with the highest similarity metric found is determined to be the target block. The block of interest is then moved to the corresponding position of the target block. In some embodiments, the similarity metric is the mean of absolute differences, the sum of absolute differences, the mean of squared differences, or the sum of squared differences, where the difference is the pixel intensity difference between the two blocks being compared. Thus, the block matching transformation is performed on each block of interest, searching for the corresponding neighborhood in the probe image, moving its position accordingly, and forming a transformed blank image. In some embodiments, this transformed blank image is used in place of the original blank image in a later removal step (i.e., step 140).

[0209] In some embodiments, a predetermined block size and a predetermined block search size are adjustable. In some embodiments, the predetermined block size used in the block matching transformation is in the range of about 1 to about 10 pixels. In other words, the block matching transformation includes a block size in the range of about 1 to 10 pixels. In some embodiments, the predetermined block search size used in the block matching transformation is in the range of about 1 to about 10 pixels. In other words, the block matching transformation includes a block search size in the range of about 1 to 10 pixels.

[0210] In some embodiments, a method for enhancing target detection includes any combination of the steps described herein in various orders. In some embodiments, steps may be omitted. Furthermore, the order of the steps may be reversed, changed, or performed simultaneously.

[0211] In at least one embodiment, the electronic-based aspects of Method 100 may be implemented in software executable by a computer comprising one or more processing units, such as a microprocessor and / or application-specific integrated circuit ("ASIC") (e.g., storage on a non-temporary computer-readable medium). In some embodiments, hardware, software, and electronic components or modules may be included. It should be noted that multiple hardware and software-based devices, as well as multiple different structural components, can thus be used to implement the embodiments.

[0212] As described above, specific embodiments have been presented herein for illustrative purposes; however, it should be understood that various modifications may be made without departing from the spirit and scope of what is provided herein. All references mentioned above are incorporated herein by reference in their entirety. [Brief explanation of the drawing]

[0213] [Figure 1A] This diagram shows a schematic of the sequential ISH / IHC and integrated ISH / IHC codetection workflows. It also shows the steps of the sequential ISH-IHC workflow. [Figure 1B] This diagram shows a schematic representation of the sequential ISH / IHC and integrated ISH / IHC codetection workflows. It also shows the steps involved in the integrated ISH / IHC codetection workflow. [Figure 2]This shows that the IHC signal of CD20 was protected by crosslinking from stress caused by formamide-based reagents in hybridization buffer. A shows formalin-fixed paraffin-embedded (FFPE) human tonsil tissue sections stained with IHC using the Leica Bond Polymer Refine Detection Kit. B shows sections with reduced IHC signaling after exposure to hybridization buffer at room temperature for 30 minutes. C shows that the IHC signal was protected by crosslinking after primary antibody exposure despite exposure to hybridization buffer. [Figure 3] This shows that the resistance of the CD20 IHC signal to protease treatment was protected by crosslinking. A shows a human tonsil tissue section in which the CD20 protein was detected by IHC staining using the Leica Bond Polymer Refine Detection Kit. B shows the IHC signal reduced after exposure to the protease enzyme at 40°C for 15 minutes. C shows the IHC signal protected by crosslinking after primary antibody despite exposure to the protease enzyme. [Figure 4] The IHC signal for CD8 was enhanced by crosslinking before hybridization buffer or protease treatment. A shows a human tonsil tissue section stained with IHC using the Leica Bond Polymer Refine Detection Kit to detect CD8. B shows the IHC signal reduced after exposure to hybridization buffer at room temperature for 30 minutes. C shows the IHC signal enhanced by crosslinking after primary antibody despite exposure to hybridization buffer. D shows a human tonsil section stained with IHC using the Leica Bond Polymer Refine Detection Kit to detect CD8. E shows the IHC signal reduced after exposure to protease enzyme at 40°C for 15 minutes. F shows the IHC signal enhanced by crosslinking after primary antibody despite exposure to protease enzyme. [Figure 5] This demonstrates that the ISH / IHC integrated co-detection workflow enables co-detection of the CD8 protein using human peptidyl prolyl isomerase B (Hs-PPIB) ISH staining. A shows a section of a human head and neck cancer sample from FFPE that has been IHC stained for CD8 using the Leica Bond Polymer Refine Detection kit. B shows ISH staining of Hs-PPIB using the RNAscope® 2.5 LS Red kit. C shows the ISH and IHC signals after the conventional procedure in which ISH and IHC are performed sequentially. D shows the enhanced ISH and IHC signals after the ISH / IHC integrated co-detection workflow. [Figure 6] This study demonstrates that crosslinking is key to improving IHC signaling in an integrated ISH / IHC co-detection workflow. A shows a section of FFPE human tonsil tissue stained with IHC for CD20 using the Leica Bond Polymer Refine Detection kit, followed by the Green chromogen. B shows a section of FFPE human tonsil tissue stained with ISH for Hs-PPIB using the RNAscope® 2.5 LS Red kit. C shows a section of FFPE human tonsil tissue pre-treated with RNAscope®, including a 15-minute incubation with protease at 40°C before IHC staining, resulting in reduced IHC signaling. D shows a section of FFPE human tonsil tissue treated with an integrated ISH / IHC co-detection workflow without a crosslinking step. E shows a section of FFPE human tonsil tissue treated with an integrated ISH / IHC co-detection workflow including a crosslinking step. [Figure 7]This shows that crosslinking at room temperature was effective when performed for 15–60 minutes. A shows sections of FFPE human gastric cancer tissue that were stained with ISH for Hs-PPIB detection and immediately followed by CD8 IHC detection using RNAscope® 2.5 LS Red and Leica Bond Polymer Refine Detection kits, respectively. B shows sections of FFPE human gastric cancer tissue that were crosslinked at room temperature for 15 minutes using the ISH / IHC integrated co-detection workflow. C shows sections of FFPE human gastric cancer tissue that were crosslinked at room temperature for 30 minutes using the ISH / IHC integrated co-detection workflow. D shows sections of FFPE human gastric cancer tissue that were crosslinked at room temperature for 60 minutes using the ISH / IHC integrated co-detection workflow. [Figure 8] This shows that the IHC signal was preserved by crosslinking at the heated temperature. A shows a section of FFPE human gastric cancer tissue that was stained with ISH for Hs-PPIB detection and then immediately subjected to CD8 IHC detection. B shows a section of FFPE human gastric cancer tissue that was crosslinked at 40°C for 15 minutes using an ISH / IHC integrated co-detection workflow. C shows a section of FFPE human gastric cancer tissue that was crosslinked at 40°C for 30 minutes using an ISH / IHC integrated co-detection workflow. D shows a section of FFPE human gastric cancer tissue that was crosslinked at 40°C for 60 minutes using an ISH / IHC integrated co-detection workflow. E shows a section of FFPE human gastric cancer tissue that was crosslinked at 60°C for 15 minutes using an ISH / IHC integrated co-detection workflow. F shows a section of FFPE human gastric cancer tissue that was crosslinked at 60°C for 30 minutes using an ISH / IHC integrated co-detection workflow. Figure G shows sections of FFPE human gastric cancer tissue crosslinked at 60°C for 60 minutes using an ISH / IHC integrated co-detection workflow. [Figure 9]This shows that the IHC signal was preserved by crosslinking at 4°C. A shows a section of FFPE human gastric cancer tissue that was stained with ISH for Hs-PPIB detection and then immediately subjected to CD8 IHC detection. B shows a section of FFPE human gastric cancer tissue that was crosslinked at room temperature for 30 minutes using an ISH / IHC integrated co-detection workflow. C shows a section of FFPE human gastric cancer tissue that was crosslinked at 4°C for 2 hours using an ISH / IHC integrated co-detection workflow. D shows a section of FFPE human gastric cancer tissue that was crosslinked overnight at 4°C using an ISH / IHC integrated co-detection workflow. [Figure 10A] A typical workflow diagram of an image processing method for reducing background signal according to one embodiment of this disclosure is shown, along with a description of the image being processed. [Figure 10B] A typical workflow diagram of an image processing method for reducing background signal according to one embodiment of this disclosure is shown, and the general steps of this method are described.

Claims

1. A method for detecting target nucleic acids and target proteins in a single biological sample, (i) Incubating the biological sample with the primary antibody, (ii) Treating the biological sample with a crosslinking agent, (iii) Treating the biological sample with a protease, (iv)Detecting the target nucleic acid by in situ hybridization, (v) The method comprising detecting the target protein by incubating the biological sample with a secondary antibody or other labeling method.

2. The method according to claim 1, wherein the target nucleic acid is RNA.

3. The method according to claim 1, wherein the target nucleic acid is DNA.

4. The step of detecting the target nucleic acid by in situ hybridization includes (i) providing one or more target probes capable of hybridizing to the target nucleic acid, (ii) To provide a signal-generating complex that can hybridize to one or more target probes, wherein the signal-generating complex comprises a nucleic acid component that can hybridize to one or more target probes, and a labeled probe. (iii) Hybridizing the target nucleic acid with one or more target probes, (iv) The method according to any one of claims 1 to 3, comprising capturing the signal-generating complex on one or more target probes, thereby capturing the signal-generating complex on the target nucleic acid.

5. The method according to claim 4, wherein each of the one or more target probes comprises a target (T) section and a label (L) section, the T section being a nucleic acid sequence complementary to the section on the target nucleic acid, the L section being a nucleic acid sequence complementary to the section on the nucleic acid component of the signal generation complex, the T section of the one or more target probes being complementary to a non-overlapping region of the target nucleic acid, and the L section of the one or more target probes being complementary to a non-overlapping region of the nucleic acid component of the generation complex.

6. The method according to any one of claims 1 to 5, further comprising providing an immunohistochemical label that can bind to the secondary antibody for detecting the target protein, or the secondary antibody is pre-labeled.

7. The method according to any one of claims 1 to 6, wherein the biological sample is a tissue sample or derived from a tissue sample.

8. The method according to any one of claims 1 to 6, wherein the biological sample is a blood sample or derived from a blood sample.

9. The method according to any one of claims 1 to 6, wherein the biological sample is a cytological sample or is derived from a cytological sample.

10. The method according to any one of claims 1 to 6, wherein the biological sample is a cultured cell or an exosome-containing sample.

11. The method according to claim 1, wherein the crosslinking agent is a fixing liquid.

12. The method according to claim 11, wherein the fixative is neutral buffered formalin.

13. The method according to claim 12, wherein the neutral buffered formalin is 10% neutral buffered formalin.

14. The method according to any one of claims 1 to 13, wherein the step of treating the biological sample with the crosslinking agent lasts for approximately 15 minutes, approximately 30 minutes, approximately 60 minutes, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, or approximately 24 hours.

15. The method according to any one of claims 1 to 13, wherein the step of treating the biological sample with the crosslinking agent is carried out at approximately 4°C, room temperature, approximately 40°C, or approximately 60°C.

16. The method according to any one of claims 1 to 13, wherein the step of treating the biological sample with the crosslinking agent is carried out at approximately 4°C for approximately 2 hours.

17. The method according to any one of claims 1 to 13, wherein the step of treating the biological sample with the crosslinking agent is carried out at approximately 4°C for approximately 16 to approximately 18 hours.

18. The method according to any one of claims 1 to 13, wherein the step of treating the biological sample with the crosslinking agent is carried out at room temperature for about 15 minutes.

19. The method according to any one of claims 1 to 13, wherein the step of treating the biological sample with the crosslinking agent is carried out at room temperature for about 30 minutes.

20. The method according to any one of claims 1 to 13, wherein the step of treating the biological sample with the crosslinking agent is carried out at room temperature for about 60 minutes.

21. The method according to any one of claims 1 to 13, wherein the step of treating the biological sample with the crosslinking agent is carried out at approximately 40°C for approximately 15 minutes.

22. The method according to any one of claims 1 to 13, wherein the step of treating the biological sample with the crosslinking agent is carried out at approximately 40°C for approximately 30 minutes.

23. The method according to any one of claims 1 to 13, wherein the step of treating the biological sample with the crosslinking agent is carried out at approximately 40°C for approximately 60 minutes.

24. The method according to any one of claims 1 to 13, wherein the step of treating the biological sample with the crosslinking agent is carried out at approximately 60°C for approximately 15 minutes.

25. The method according to any one of claims 1 to 13, wherein the step of treating the biological sample with the crosslinking agent is carried out at approximately 60°C for approximately 30 minutes.

26. The method according to any one of claims 1 to 13, wherein the step of treating the biological sample with the crosslinking agent is carried out at approximately 60°C for approximately 60 minutes.

27. The method according to any one of claims 1 to 26, wherein the method is used to map the spatial configuration in a composite tissue.

28. The method according to claim 27, wherein the composite tissue is tumor tissue.

29. The method according to any one of claims 1 to 26, used for detecting denatured gene expression in a biological sample from a diseased model.

30. A method according to any one of claims 1 to 26, used for validating a novel antibody.

Images