Method and kit for detecting target nucleic acids by in situ hybridization

Abstract

1. A method for detecting a target nucleic acid in a cell, such as a small non-coding RNA (sncRNA), microRNA (miRNA), small interfering RNA (siRNA), PIWI-interacting RNA (piRNA), or antisense oligonucleotide (ASO) molecule, comprising a post-fixation step with an aldehyde-containing fixative after an initial fixation step and before in situ hybridization.

Description

Detailed Description of the Invention

[0001] 〔Technical Field〕 In some embodiments, and without limitation, methods for preparing a biological sample for in situ hybridization for detecting, for example, small non-coding RNAs (sncRNAs), microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), small interfering RNAs (siRNAs) or antisense oligonucleotide (ASO) molecules are provided herein. Further, methods for detecting target nucleic acids in cells are provided. Also provided herein are kits for carrying out the above methods and biological samples prepared by the above methods in certain embodiments.

[0002] 〔Background Art〕 In situ hybridization (ISH) is a technique that enables precise detection of specific segments of nucleic acids in histological sections. The principle underlying ISH is that when nucleic acids are appropriately preserved in histological specimens, the preserved nucleic acids can be detected by applying complementary nucleic acid strands to which reporter molecules are bound. The target nucleic acid can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0003] Cells contain various types of RNA, including (i) messenger RNA (mRNA) that encodes the amino acid sequence of polypeptides, (ii) transfer RNA (tRNA) that guides amino acids to ribosomes during translation, (iii) ribosomal RNA (rRNA) that constitutes ribosomes, the organelles that translate mRNA, and (iv) sncRNAs involved in various RNA processing processes. In particular, sncRNAs, miRNAs, siRNAs, piRNAs, and ASOs are of high importance in research, diagnostics, and drug development (see, for example, Hanna et al., Frontiers in Genetics, 10, 1-6, 2019, and Watts et al., Journal of Pathology, 226(2), 365-379, 2012). There is a need for methods that demonstrate improved detection sensitivity of RNA, especially small RNAs, within the tissue microenvironment, such as those measured by RNA ISH technology.

[0004] Despite the crucial role of small nucleic acids in diverse biological processes, their detection presents challenges. Tissue preservation for histopathology relies on crosslinking of proteins and nucleic acids using chemical fixatives. This fixation strategy is compatible with current RNA ISH techniques for detecting long messenger RNA (mRNA) species. However, this method improperly crosslinks small nucleic acids, leading to their divergence and dispersion from the tissue, resulting in reduced detection by ISH assays. Therefore, novel compositions and methods are needed for the detection of a wider range of nucleic acid molecules by ISH.

[0005] [Summary of the Invention] In one embodiment, the Specified provides a method for preparing a biological sample for in situ hybridization, comprising: i) a first fixation step of fixing the biological sample with a drug; and ii) a post-fixation step of fixing the biological sample with an aldehyde-containing fixative, which occurs after the first fixation step and before in situ hybridization.

[0006] In some embodiments, this method can be used to prepare biological samples for RNA in situ hybridization. In some embodiments, this method can be used to prepare biological samples for DNA in situ hybridization.

[0007] In some embodiments, the in situ hybridization method of this disclosure can be used to detect nucleic acids containing fewer than 100 nucleotides. In some embodiments, the in situ hybridization method of RNA can be used to detect RNA containing fewer than 50 nucleotides. In some embodiments, the in situ hybridization method of RNA can be used to detect RNA containing 15 to 40 nucleotides.

[0008] In some embodiments, the RNA in situ hybridization method of this disclosure can be used to detect small non-coding RNA (sncRNA). In some embodiments, the RNA in situ hybridization method can be used to detect microRNA (miRNA), small interfering RNA (siRNA) or PIWI-interacting RNA (piRNA), or antisense oligonucleotide (ASO) molecules. In some embodiments, the RNA in situ hybridization method can be used to detect endogenous RNA. In some embodiments, the RNA in situ hybridization method can be used to detect exogenous RNA.

[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 other embodiments, the biological sample is a cytological sample or derived from a cytological sample. In other embodiments, the biological sample is cultured cells. In other embodiments, the biological sample is an exosome.

[0010] In some embodiments, the agent in the first fixation step is selected from the group consisting of formaldehyde, glutaraldehyde, alcohol (methanol, ethanol), and acetone.

[0011] In other embodiments, the aldehyde-containing fixative in the post-fixation step is selected from the group consisting of formaldehyde or glutaraldehyde.

[0012] In some embodiments, the aldehyde-containing fixative contains about 5% to about 50% formaldehyde. In certain embodiments, the aldehyde-containing fixative contains less than about 12% formaldehyde. In some embodiments, the post-fixation step includes fixing the biological sample with the aldehyde-containing fixative for more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 hours.

[0013] In certain embodiments, the aldehyde-containing fixative contains at least about 12% formaldehyde, and the post-fixation step includes fixing the biological sample with the aldehyde-containing fixative for about 2 hours. In certain embodiments, the aldehyde-containing fixative contains about 12% to about 37% formaldehyde, and the post-fixation step includes fixing the biological sample with the aldehyde-containing fixative for about 2 hours.

[0014] In some embodiments, biological samples are prepared according to the method described above.

[0015] In another embodiment, embodiments of the present disclosure include a method for detecting a target nucleic acid in a cell, comprising: (i) preparing a biological sample; (ii) providing at least one set of one or more target probes that can hybridize to the target nucleic acid; (iii) providing a signal-generating complex that can hybridize to the set of one or more target probes, wherein the signal-generating complex comprises a nucleic acid component that can hybridize to the set of one or more target probes, and a labeled probe; (iv) hybridizing the target nucleic acid to the set of one or more target probes; and (v) capturing the signal-generating complex on the set of one or more target probes, thereby capturing the signal-generating complex on the target nucleic acid.

[0016] In certain embodiments, each target probe(s) includes a target (T) section and a label (L) section. In some 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 another specific embodiment, the T section of one or more target probes(s) is complementary to a non-overlapping region of the target nucleic acid, and the L section of one or more target probes(s) is complementary to a non-overlapping region of the nucleic acid component of the signal-generating complex.

[0017] In some embodiments, the target nucleic acid contains fewer than approximately 100 nucleotides. In other embodiments, the target nucleic acid is RNA containing fewer than approximately 50 nucleotides. In other embodiments, the target nucleic acid is RNA containing approximately 15 to approximately 40 nucleotides.

[0018] In some embodiments, the target nucleic acid is sncRNA. In some embodiments, the target nucleic acid is miRNA, siRNA, piRNA, or ASO. In some embodiments, the target nucleic acid is endogenous nucleic acid. In other embodiments, the target nucleic acid is exogenous nucleic acid.

[0019] In some embodiments, the method is used to identify tissue or cell type. In other embodiments, the method is used to determine developmental stage. In other embodiments, the method is used to characterize adult tissue. In other embodiments, the method is used to diagnose disease or disorder based on the expression of one or more denatured small RNAs or the presence of pathogen-derived small RNAs. In other embodiments, the method is used to monitor or determine the effectiveness of small RNA or oligonucleotide-based therapies.

[0020] In another embodiment, embodiments of the present disclosure include a method for storing a target nucleic acid in a pre-fixed sample for in situ detection of the target nucleic acid, comprising applying an aldehyde-containing fixative to the sample before performing an in situ hybridization detection assay using a set of one or more probes that hybridize to the target nucleic acid.

[0021] In another embodiment, this specification provides an in situ detection method for a target nucleic acid in a pre-fixed sample, comprising: (i) applying an aldehyde-containing fixative to the sample; and (ii) performing an in situ hybridization detection assay using a set of one or more probes that hybridize to the target nucleic acid.

[0022] In some embodiments, the target nucleic acid contains fewer than approximately 100 nucleotides. In other embodiments, the target nucleic acid is RNA containing fewer than approximately 50 nucleotides. In other embodiments, the target nucleic acid is RNA containing approximately 15 to approximately 40 nucleotides.

[0023] 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 other embodiments, the biological sample is a cytological sample or derived from a cytological sample. In other embodiments, the biological sample is cultured cells. In other embodiments, the biological sample is an exosome.

[0024] In other embodiments, the aldehyde-containing fixative in the post-fixation step includes formaldehyde or glutaraldehyde.

[0025] In some embodiments, the aldehyde-containing fixative contains about 5% to about 50% formaldehyde. In certain embodiments, the aldehyde-containing fixative contains less than about 12% formaldehyde, and the post-fixation step includes fixing the biological sample with the aldehyde-containing fixative for more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 hours. In certain embodiments, the aldehyde-containing fixative contains at least about 12% formaldehyde, and the post-fixation step includes fixing the biological sample with the aldehyde-containing fixative for about 2 hours. In certain embodiments, the aldehyde-containing fixative contains about 12% to about 37% formaldehyde, and the post-fixation step includes fixing the biological sample with the aldehyde-containing fixative for about 2 hours.

[0026] In some embodiments, the probe set comprises one or more target probes capable of hybridizing to the target nucleic acid, and the method further comprises (a) 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; (b) hybridizing the target nucleic acid to the set of one or more target probes; and (c) capturing the signal-generating complex on the set of one or more target probes, thereby capturing the signal-generating complex on the target nucleic acid.

[0027] In some embodiments, each of the target probe(s) includes 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 generation complex. In certain embodiments, the T sections of one or more target probe(s) are complementary to non-overlapping regions of the target nucleic acid, and the L sections of one or more target probe(s) are complementary to non-overlapping regions of the nucleic acid component of the generation complex.

[0028] In another aspect, a kit for in situ detection of a target nucleic acid in a cell includes (i) an agent for fixing a biological sample containing the cell, (ii) an aldehyde-containing fixative for fixing the biological sample, (iii) an agent for performing in situ hybridization, and (iv) an instruction manual indicating that the aldehyde-containing fixative is used after the agent of element (i). In some embodiments, a kit for in situ detection of a target nucleic acid in a cell includes an aldehyde-containing fixative for fixing the biological sample and an agent for performing in situ hybridization.

[0029] In some embodiments, the agent of element (i) is selected from the group consisting of formaldehyde, glutaraldehyde, alcohol (methanol, ethanol), and acetone. In other embodiments, the aldehyde-containing fixative includes formaldehyde or glutaraldehyde.

[0030] In some embodiments, the aldehyde-containing fixative includes from about 5% to about 50% formaldehyde. In certain embodiments, the aldehyde-containing fixative includes at least about 12% formaldehyde. In certain embodiments, the aldehyde-containing fixative includes from about 12% to about 37% formaldehyde.

[0031] In some embodiments, the agent performing in situ hybridization includes at least one set of one or more target probes capable of hybridizing to the target nucleic acid, and a signal-generating complex capable of hybridizing to the set of one or more target probes. In certain embodiments, the signal-generating complex includes a nucleic acid component capable of hybridizing to the set of one or more target probes, and a labeled probe.

[0032] In some embodiments, each target probe(s) includes 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 sections of one or more target probe(s) are complementary to non-overlapping regions of the target nucleic acid, and the L sections of one or more target probe(s) are complementary to non-overlapping regions of the nucleic acid component of the signal-generating complex.

[0033] In some embodiments, the kit further includes tools for obtaining a biological sample. 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. In certain embodiments, the biological sample is cultured cells. In certain embodiments, the biological sample is an exosome.

[0034] In some embodiments, the target nucleic acid contains fewer than approximately 100 nucleotides. In other embodiments, the target nucleic acid is RNA containing fewer than 50 nucleotides. In other embodiments, the target nucleic acid is RNA containing approximately 15 to approximately 40 nucleotides. In some embodiments, the target nucleic acid is sncRNA. In other embodiments, the target nucleic acid is miRNA, siRNA, piRNA, or ASO. In other embodiments, the target nucleic acid is endogenous or exogenous.

[0035] In some embodiments, the kit is used to identify tissue or cell type. In other embodiments, the kit is used to determine developmental stage. In other embodiments, the kit is used to characterize adult tissue. In other embodiments, the kit is used to diagnose disease or disorder based on the expression of one or more denatured small RNAs or the presence of pathogen-derived small RNAs. In other embodiments, the method is used to monitor or determine the effectiveness of small RNA-based therapies.

[0036] [Brief explanation of the drawing] [Figure 1] This shows the incorporation of post-fixation steps in a standard RNA ISH workflow. Post-fixation steps that are not present in a standard RNA ISH workflow are labeled with a double-bordered box.

[0037] [Figure 2A] This figure shows that post-fixation with formaldehyde at or above 12% minimized miRNA loss and improved detection sensitivity by RNAscope® ISH. Mouse formalin-fixed paraffin-embedded tissue sections were left untreated or post-fixed with formaldehyde concentrations ranging from 12% to 37%. Two miRNAs were detected using RNAscope® 2.5 High Definition (HD)-Red Assay according to established conditions for formalin-fixed paraffin-embedded tissue processing. The ISH signal appeared as spots, and hematoxylin stained individual cell nuclei. This figure shows the detection of miR-1a-3p, which is abundantly expressed in the heart, after the post-fixation process.

[0038] [Figure 2B] This figure shows that post-fixation with formaldehyde at or above 12% minimized miRNA loss and improved detection sensitivity by RNAscope® ISH. Mouse formalin-fixed paraffin-embedded tissue sections were left untreated or post-fixed with formaldehyde concentrations ranging from 12% to 37%. Two miRNAs were detected using RNAscope® 2.5 High Definition (HD)-Red Assay according to established conditions for formalin-fixed paraffin-embedded tissue processing. The ISH signal appeared as spots, and hematoxylin stained individual cell nuclei. This figure shows the detection of miR-132-3p, which is abundantly expressed in the brain.

[0039] [Figure 3A] This figure shows that post-fixation with 10% neutral buffered formalin (NBF) containing approximately 4% formaldehyde minimized miRNA loss and improved detection sensitivity by RNAscope® ISH. Mouse formalin-fixed paraffin-embedded tissue sections were either left untreated or post-fixed with 10% NBF. Multiple miRNAs were detected using RNAscope® 2.5 High Definition (HD)-Red Assay according to established conditions for formalin-fixed paraffin-embedded tissue processing. ISH signals appeared as spots, and hematoxylin stained individual cell nuclei. This figure shows the detection of miR-1a-3p, which is abundantly expressed in the heart, after the post-fixation process.

[0040] [Figure 3B] This figure shows that post-fixation with 10% neutral buffered formalin (NBF) containing approximately 4% formaldehyde minimized miRNA loss and improved detection sensitivity by RNAscope® ISH. Mouse formalin-fixed paraffin-embedded tissue sections were either left untreated or post-fixed with 10% NBF. Multiple miRNAs were detected using RNAscope® 2.5 High Definition (HD)-Red Assay according to established conditions for formalin-fixed paraffin-embedded tissue processing. ISH signals appeared as spots, and hematoxylin stained individual cell nuclei. This figure shows the detection of miR-132-3p, which is abundantly expressed in the brain, after the post-fixation process.

[0041] [Figure 3C] This figure shows that post-fixation with 10% neutral buffered formalin (NBF) containing approximately 4% formaldehyde minimized miRNA loss and improved detection sensitivity by RNAscope® ISH. Mouse formalin-fixed paraffin-embedded tissue sections were either left untreated or post-fixed with 10% NBF. Multiple miRNAs were detected using RNAscope® 2.5 High Definition (HD)-Red Assay according to established conditions for formalin-fixed paraffin-embedded tissue processing. ISH signals appeared as spots, and hematoxylin stained individual cell nuclei. This figure shows the detection of miR-122-5p, which is abundantly expressed in the liver, after the post-fixation process.

[0042] [Figure 3D] This figure shows that post-fixation with 10% neutral buffered formalin (NBF) containing approximately 4% formaldehyde minimized miRNA loss and improved detection sensitivity by RNAscope® ISH. Mouse formalin-fixed paraffin-embedded tissue sections were either left untreated or post-fixed with 10% NBF. Multiple miRNAs were detected using RNAscope® 2.5 High Definition (HD)-Red Assay according to established conditions for formalin-fixed paraffin-embedded tissue processing. ISH signals appeared as spots, and hematoxylin stained individual cell nuclei. This figure shows the detection of eccentrically expressed let-7 after the post-fixation process.

[0043] [Figure 4A] This shows that the post-fixation workflow was fully suitable for detecting long RNA species. Human and mouse formalin-fixed paraffin-embedded tissues were either left untreated or post-fixed with 10% NBF and then processed using the general-purpose ISH workflow outlined in Figure 1. This figure shows the detection of human TATA box-binding protein (TBP) mRNA in human cutaneous A431 cells using RNAscope® 2.5 High Definition (HD)-Red Assay.

[0044] [Figure 4B] This shows that the post-fixation workflow was fully suitable for detecting long RNA species. Human and mouse formalin-fixed paraffin-embedded tissues were either left untreated or post-fixed with 10% NBF and then processed using the general-purpose ISH workflow outlined in Figure 1. This figure shows the detection of mouse polymerase RNA II polypeptide A (POLR2A) mRNA in mouse intestinal samples.

[0045] [Figure 4C] This shows that the post-fixation workflow was perfectly suited for detecting long RNA species. Human and mouse formalin-fixed paraffin-embedded tissues were either left untreated or post-fixed with 10% NBF and then processed using the general-purpose ISH workflow outlined in Figure 1. This figure shows the detection of mouse polymerase RNA II polypeptide A (POLR2A) mRNA in brain tissue.

[0046] [Modes for carrying out the invention] The method provided herein is based in part on the unexpected discovery that a post-fixation step, performed after the first fixation step but before in situ hybridization, provides better retention of low-molecular-weight target nucleic acids, such as low-molecular-weight RNA species in biological samples, improves the detection sensitivity of target nucleic acids, such as low-molecular-weight RNA species, and furthermore, that the method has complete compatibility with long-chain nucleic acid species, such as long-chain RNA species. The method provided herein preserves low-molecular-weight nucleic acid molecules in the natural tissue microenvironment and provides higher detection sensitivity. To minimize loss, pre-fixed biological samples are post-fixed with an aldehyde-containing fixative, and then the samples are processed for an ISH assay (see Figure 1), thereby ensuring the preservation of low-molecular-weight nucleic acids within the biological sample. This practical and reliable strategy has complete compatibility with standard ISH assays (see Figures 2, 3, and 4), enhances the detection sensitivity of low-molecular-weight nucleic acids, and enables spatial identification of target nucleic acids such as sncRNA, miRNA, siRNA, piRNA, and ASO.

[0047] 5.1 Definition As used herein, the terms “fixation” or “to fix” refer to the fixation of a biological specimen in an in situ hybridization process, meaning a procedure to protect the biological specimen from disintegration, for example, autolysis or decomposition. This procedure may halt any ongoing biochemical reactions and further improve the mechanical strength or stability of the treated tissue.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] As used herein, the term "sample" refers to a substance or mixture of substances containing one or more components of interest. 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, cell cultures, cell lines, tissues, oral tissues, gastrointestinal tissues, organs, organelles, 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.

[0053] As used herein, the term “probe” refers to a capture substance directed towards a specific target mRNA sequence. Therefore, each probe in a set of probes has its own target mRNA sequence. In some embodiments, the probes provided herein are “nucleic acid probes” or “oligonucleotide probes,” which refer to nucleic acids that can bind to a target nucleic acid having a complementary sequence, such as the mRNA biomarker provided herein, typically 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.

[0054] As used herein, the term “antisense oligonucleotide (ASO) molecule” or “ASO” means a short, single-stranded molecule that interacts with messenger RNA to prevent translation of a target gene.

[0055] As used herein, the term "endogenous" means a substance that originates within a living organism. As used herein, the term "exogenous" means a substance that originates outside a living organism.

[0056] As used in this disclosure and claims, the singular forms "a," "an," and "the" refer to multiple subjects unless the context explicitly indicates otherwise.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 5.2 Post-fixation for in situ hybridization Fixation is widely used to preserve biological samples from decay, such as autolysis or decomposition. Fixation can halt any ongoing biochemical reactions and improve the mechanical strength or stability of the treated tissue. However, it has long been known that standard fixation processes often compromise the quality of nucleic acids, DNA, or RNA in biological samples. With the increasing use of molecular testing in the clinical world, such limitations may be unnecessarily restricting the use of these tests. Therefore, in this regard, there is a need to optimize the detection of DNA and RNA in tissue structures.

[0061] Attempts to catalog RNA species, particularly small RNAs, heavily rely on microarrays, quantitative polymerase chain reaction (qPCR), and sequencing techniques. While these methods provide bulk expression levels, detailed spatial expression information for specific miRNAs is lost in the process. This highlights the need to detect and catalog miRNAs based on tissue or cell type-specific expression patterns. In situ hybridization (ISH) is a commonly used method for visualizing the expression of genes of interest within the cellular microenvironment. While many ISH methods are available for detecting messenger RNA (mRNA), the detection of small nucleic acids remains a challenge in reproducibly detecting and quantifying their expression.

[0062] Conventional tissue fixation methods using 10% formalin provide optimal preservation for long-chain nucleic acid species such as mRNA, but are insufficient for fixing low-molecular-weight nucleic acid species such as miRNA, siRNA, piRNA, and ASO. Recently, a fixation method has been introduced that prevents the loss of miRNA from histological samples using the crosslinking agent formaldehyde, followed by the chemical fixative 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). When visualized by RNA ISH based on hapten-labeled probes and colorimetric antibodies, miRNAs in the tissue microenvironment are preserved (Pena et al., Nature Methods, 6(2), 139-141, 2010). Although this strategy is useful, it presents several challenges. Firstly, the crosslinking process with EDC is highly sensitive to pH, resulting in various miRNA retention profiles and increasing potential variability between assays. Secondly, because the reagents are unstable, EDC must be freshly prepared and mixed with stabilizers to maintain its fixative properties (Renwick, et al., The Journal of Clinical Investigation, 123(6), 2694-2702, 2013). Therefore, these limitations hinder the practical application of these methods for detecting small RNA species, especially in large-scale projects. Consequently, a more practical and stable method is needed that minimizes the loss of small nucleic acids in ISH assays and improves the detection sensitivity of small nucleic acids.

[0063] Accordingly, embodiments of this disclosure encompass a versatile and reliable method for preserving low-molecular-weight nucleic acid molecules within biological samples, which is fully compatible with established ISH assays. Nucleic acid species retention is achieved by exposing a pre-fixed biological sample to an aldehyde-containing fixative prior to ISH. This strategy eliminates the need to prepare unstable chemical compounds, provides a reliable method for preserving nucleic acids within tissues, and leads to improved detection sensitivity.

[0064] The method of the present disclosure includes applying an aldehyde-containing fixative to a pre-fixed sample. In some embodiments, the present invention provides a method for preserving a target nucleic acid in a pre-fixed sample for in situ detection of the target nucleic acid, the method comprising applying an aldehyde-containing fixative to the sample before performing an in situ hybridization detection assay using a set of one or more probes that hybridize to the target nucleic acid.

[0065] In some embodiments, the sample was pre-fixed by chemical fixation using fixative(s). In some embodiments, the sample was pre-fixed by immersion in fixative(s), perfusion of biological sample with fixative(s), or application of vaporized fixative(s). In other embodiments, the sample was pre-fixed by physical fixation, such as heating, microwave exposure, cryopreservation, or freeze-drying.

[0066] In some embodiments, the sample was pre-fixed with a coagulating fixative(s). In other embodiments, the sample was pre-fixed with a non-coagulating fixative(s). In some embodiments, the sample was pre-fixed with a fixative(s) having denaturing properties that remove water from the biological sample. In other embodiments, the sample was pre-fixed with a crosslinking fixative(s) that forms chemical bonds between molecules of the biological sample. In some embodiments, the sample was pre-fixed with an additive fixative(s) (i.e., the fixative(s) becomes part of the biological sample that is processed in subsequent steps of the histological protocol). In other embodiments, the sample was pre-fixed with a non-additive fixative(s) (i.e., once the fixation is performed and completed, the fixative(s) is removed from the biological sample in subsequent steps of the histological protocol).

[0067] In one embodiment, the sample was pre-fixed with ethanol. In one embodiment, the sample was pre-fixed with methanol. In one embodiment, the sample was pre-fixed with acetone. In one embodiment, the sample was pre-fixed with acetic acid. In one embodiment, the sample was pre-fixed with zinc chloride. In one embodiment, the sample was pre-fixed with zinc sulfate. In one embodiment, the sample was pre-fixed with picric acid. In one embodiment, the sample was pre-fixed with formaldehyde. In one embodiment, the sample was pre-fixed with glutaraldehyde. In one embodiment, the sample was pre-fixed with osmium tetroxide. In some specific embodiments, the sample was pre-fixed with carbodiimide. In some specific embodiments, the sample was pre-fixed with diimide esters. In one embodiment, the sample was pre-fixed with chloro-s-triazide (cyanuryl chloride). In some specific embodiments, the sample was pre-fixed with diisocyanate. In one embodiment, the sample was pre-fixed with diethyl pyrocarbonate (DPC). In some specific embodiments, the sample was pre-fixed with maleimide. In one embodiment, the sample was pre-fixed with benzoquinone. In one embodiment, the sample was pre-fixed with mercury chloride. In another embodiment, the sample was pre-fixed with potassium dichromate. In yet another embodiment, the sample was pre-fixed with potassium permanganate. In yet another embodiment, the sample was pre-fixed with chromic acid.

[0068] In one embodiment, the sample was pre-fixed with Bouin fixative, which is a solution of picric acid, formaldehyde, and acetic acid. In another embodiment, the sample was pre-fixed with Clarke fixative, which is a solution of ethanol and acetic acid. In yet another embodiment, the sample was pre-fixed with Carnoy fixative, which is a solution of ethanol, chloroform, and acetic acid. In yet another embodiment, the sample was pre-fixed with a mixed solution of formaldehyde and glutaraldehyde. In yet another embodiment, the sample was pre-fixed with FAA, which is a solution of ethanol, acetic acid, and formaldehyde. In yet another embodiment, the sample was pre-fixed with periodate-lysine-paraformaldehyde (PLP), which is a solution of paraformaldehyde, L-lysine, and INaO4. In yet another embodiment, the sample was pre-fixed with phosphate-buffered formalin (PBF). In yet another embodiment, the sample was pre-fixed with formal-calcium, which is a solution of formaldehyde and calcium chloride. In one embodiment, the sample was pre-fixed with formalin-physiological saline solution, which is a solution of formaldehyde and sodium chloride. In another embodiment, the sample was pre-fixed with zinc formalin solution, which is a solution of formaldehyde and zinc sulfate. In another embodiment, the sample was pre-fixed with Zenker fixative, which is a solution of mercury chloride, potassium dichromate, and glacial acetic acid. In yet another embodiment, the sample was pre-fixed with Helly fixative, which is a solution of formaldehyde, potassium dichromate, sodium sulfate, and mercury chloride. In yet another embodiment, the sample was pre-fixed with B-5 fixative, c mercuric chloride, and sodium acetate. In yet another embodiment, the sample was pre-fixed with Hollande fixative, which is a solution of formaldehyde, copper acetate, picric acid, and acetic acid. In yet another embodiment, the sample was pre-fixed with Gendre solution, which is a solution of formaldehyde, ethanol, picric acid, and glacial acetic acid. In one embodiment, the sample was pre-fixed with Methacarn fixative, which is a solution of methanol, chloroform, and glacial acetic acid.In one embodiment, the sample was pre-fixed with alcohol formalin, which is a solution of formaldehyde, ethanol, and calcium acetate. In another embodiment, the sample was pre-fixed with formalic acid alcohol, which is a solution of formaldehyde, glacial acetic acid, and ethanol.

[0069] In some embodiments, the sample was pre-fixed with a mixture of two, three, four, or five or more fixatives selected from a list including ethanol, methanol, acetone, acetic acid, zinc chloride, zinc sulfate, picric acid, osmium tetroxide, formaldehyde, glutaraldehyde, carbodiimide, diimide esters, chloro-s-triazide (cyanuric chloride), diisocyanate, diethyl pyrocarbonate (DPC), maleimide, benzoquinone, mercury chloride, potassium dichromate, potassium permanganate, and chromic acid.

[0070] In some embodiments, the sample was pre-fixed with two or more fixatives applied sequentially, though not simultaneously. The two or more fixatives were selected from a list including ethanol, methanol, acetone, acetic acid, zinc chloride, zinc sulfate, picric acid, osmium tetroxide, formaldehyde, glutaraldehyde, carbodiimide, diimide esters, chloro-s-triazide (cyanuric chloride), diisocyanate, diethyl pyrocarbonate (DPC), maleimide, benzoquinone, mercury chloride, potassium dichromate, potassium permanganate, and chromic acid.

[0071] In some embodiments, the sample was pre-fixed with a fixative(s) suitable for preserving nucleic acids. In one embodiment, the fixative was FineFix (see Kothmaier et al., Arch. Pathol. Lab. Med. 135:744-752, 2011). In another embodiment, the fixative was Glyo-fix (see Lykidis et al., Nucleic Acids Res. 35:e85, 2007). In yet another embodiment, the fixative was Histochoice (see Vince et al., Anal. Cell. Pathol. 15:119-129, 1997). In yet another embodiment, the fixative was HOPE (see Kothmaier et al., Arch. Pathol. Lab. Med. 135:744-752, 2011). In one embodiment, the fixative is Neo-Fix (see Paavilainen et al., Histochem.Cytochem.:Official J.Histochem.Soc.58:237-246, 2010). In one embodiment, the fixative is PAXgene Tissue System (see Nietner et al., Int.J.Patrol.461:259-269, 2012). In one embodiment, the fixative is RCL2 (see van Essen et al., Clin.Patrol.63:1090-1094, 2010). In one embodiment, the fixative is Streck's Tissue Fixative (see Burns et al., Histochem.Cytochem.57:257-264, 2009). In one embodiment, the fixative is UMFIX (see Nadji et al., Appl. Immunohistochem. Mol. Morphol. 13:277-282, 2005). In one embodiment, the fixative is Z7 (see Lykidis et al., Nucleic Acids Res. 35:e85, 2007). In one embodiment, the fixative is ZBF (see Paavilainen et al., Histochem. Cytochem.:Official J. Histochem. Soc. 58:237-246, 2010).

[0072] A method for preparing a biological sample for in situ hybridization provided herein comprises applying an aldehyde-containing fixative(s) to a pre-fixed sample prior to in situ hybridization. In one embodiment, the aldehyde-containing fixative comprises formaldehyde. In one embodiment, the aldehyde-containing fixative comprises glutaraldehyde. In one embodiment, the aldehyde-containing fixative comprises Bouin fixative, which is a solution of picric acid, formaldehyde, and acetic acid. In one embodiment, the aldehyde-containing fixative comprises a mixture of formaldehyde and glutaraldehyde. In one embodiment, the aldehyde-containing fixative comprises FAA, which is a solution of ethanol, acetic acid, and formaldehyde. In one embodiment, the aldehyde-containing fixative comprises periodate-lysine-paraformaldehyde (PLP), which is a solution of paraformaldehyde, L-lysine, and INaO4. In one embodiment, the aldehyde-containing fixative contains phosphate-buffered formalin (PBF). In one embodiment, the aldehyde-containing fixative contains formal calcium, which is a solution of formaldehyde and calcium chloride. In one embodiment, the aldehyde-containing fixative contains formal saline solution, which is a solution of formaldehyde and sodium chloride. In one embodiment, the aldehyde-containing fixative contains zinc formalin, which is a solution of formaldehyde and zinc sulfate. In one embodiment, the aldehyde-containing fixative contains Helly fixative, which is a solution of formaldehyde, potassium dichromate, sodium sulfate, and mercury chloride. In one embodiment, the aldehyde-containing fixative contains Hollande fixative, which is a solution of formaldehyde, copper acetate, picric acid, and acetic acid. In one embodiment, the aldehyde-containing fixative contains Gendre solution, which is a solution of formaldehyde, ethanol, picric acid, and glacial acetic acid. In one embodiment, the aldehyde-containing fixative is an alcohol-formalin solution of formaldehyde, ethanol, and calcium acetate. In another embodiment, the aldehyde-containing fixative is a formalic acid solution of formaldehyde, glacial acetic acid, and ethanol.In one embodiment, the aldehyde-containing fixative comprises a mixture of fixatives, where at least one of the fixatives in the mixture is formaldehyde or glutaraldehyde. In another embodiment, the aldehyde-containing fixative comprises fixatives that are not used simultaneously but are used sequentially, where at least one of the fixatives is formaldehyde or glutaraldehyde.

[0073] In some embodiments, the aldehyde-containing fixative contains about 5% to about 50% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 10% to about 40% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 12% to about 37% formaldehyde.

[0074] In some embodiments, the aldehyde-containing fixative contains about 5% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 6% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 7% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 8% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 9% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 10% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 11% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 12% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 13% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 14% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 15% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 16% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 17% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 18% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 19% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 20% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 30% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 35% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 40% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 50% formaldehyde.

[0075] In some embodiments, the sample is treated with an aldehyde-containing fixative for about 2 to 30 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 2 to 25 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 2 to 20 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 2 to 18 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 2 to 15 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 2 to 10 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 2 to 5 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 2 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 3 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 4 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 5 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 6 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 7 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 8 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 9 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 10 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 11 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 12 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 13 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 14 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 15 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 16 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 17 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 18 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 19 hours. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 20 hours.

[0076] In some embodiments, the sample is treated with formaldehyde for about 2 to 30 hours. In some embodiments, the sample is treated with formaldehyde for about 2 to 25 hours. In some embodiments, the sample is treated with formaldehyde for about 2 to 20 hours. In some embodiments, the sample is treated with formaldehyde for about 2 to 18 hours. In some embodiments, the sample is treated with formaldehyde for about 2 to 15 hours. In some embodiments, the sample is treated with formaldehyde for about 2 to 10 hours. In some embodiments, the sample is treated with formaldehyde for about 2 to 5 hours. In some embodiments, the sample is treated with formaldehyde for about 2 hours. In some embodiments, the sample is treated with formaldehyde for about 3 hours. In some embodiments, the sample is treated with formaldehyde for about 4 hours. In some embodiments, the sample is treated with formaldehyde for about 5 hours. In some embodiments, the sample is treated with formaldehyde for about 6 hours. In some embodiments, the sample is treated with formaldehyde for about 7 hours. In some embodiments, the sample is treated with formaldehyde for about 8 hours. In some embodiments, the sample is treated with formaldehyde for about 9 hours. In some embodiments, the sample is treated with formaldehyde for about 10 hours. In some embodiments, the sample is treated with formaldehyde for about 11 hours. In some embodiments, the sample is treated with formaldehyde for about 12 hours. In some embodiments, the sample is treated with formaldehyde for about 13 hours. In some embodiments, the sample is treated with formaldehyde for about 14 hours. In some embodiments, the sample is treated with formaldehyde for about 15 hours. In some embodiments, the sample is treated with formaldehyde for about 16 hours. In some embodiments, the sample is treated with formaldehyde for about 17 hours. In some embodiments, the sample is treated with formaldehyde for about 18 hours. In some embodiments, the sample is treated with formaldehyde for about 19 hours. In some embodiments, the sample is treated with formaldehyde for about 20 hours.

[0077] In some specific embodiments, the sample is treated with an aldehyde-containing fixative containing less than about 12% formaldehyde for more than about 15 hours. In some specific embodiments, the sample is treated with an aldehyde-containing fixative containing less than about 12% formaldehyde for about 18 hours. In some specific embodiments, the sample is treated with an aldehyde-containing fixative containing less than about 12% formaldehyde for about 19 hours. In some specific embodiments, the sample is treated with an aldehyde-containing fixative containing less than about 12% formaldehyde for about 20 hours. In some specific embodiments, the sample is treated with an aldehyde-containing fixative containing less than about 12% formaldehyde for about 25 hours.

[0078] In other specific embodiments, the sample is treated with an aldehyde-containing fixative containing about 12% or more formaldehyde for less than 10 hours. In other specific embodiments, the sample is treated with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 10 hours. In other specific embodiments, the sample is treated with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 9 hours. In other specific embodiments, the sample is treated with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 8 hours. In other specific embodiments, the sample is treated with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 7 hours. In other specific embodiments, the sample is treated with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 6 hours. In other specific embodiments, the sample is treated with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 5 hours. In other specific embodiments, the sample is treated with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 4 hours. In other specific embodiments, the sample is treated with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 3 hours. In other specific embodiments, the sample is treated with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 2 hours.

[0079] In some specific embodiments, the aldehyde-containing fixative is in a buffer solution with a pH of approximately 6. In some specific embodiments, the aldehyde-containing fixative is in a buffer solution with a pH of approximately 6.5. In some specific embodiments, the aldehyde-containing fixative is in a buffer solution with a pH of approximately 7. In some specific embodiments, the aldehyde-containing fixative is in a buffer solution with a pH of approximately 7.5. In some specific embodiments, the aldehyde-containing fixative is in a buffer solution with a pH of approximately 8. In some specific embodiments, the aldehyde-containing fixative is in a buffer solution with a pH of approximately 8.5. In some specific embodiments, the aldehyde-containing fixative is in a buffer solution with a pH of approximately 9.

[0080] In some embodiments, fixing is carried out at a temperature of approximately 4 to 50°C. In some embodiments, fixing is carried out at a temperature of approximately 4°C. In some embodiments, fixing is carried out at a temperature of approximately 10°C. In some embodiments, fixing is carried out at a temperature of approximately 20°C. In some embodiments, fixing is carried out at a temperature of approximately 25°C. In some embodiments, fixing is carried out at a temperature of approximately 30°C. In some embodiments, fixing is carried out at a temperature of approximately 40°C. In some embodiments, fixing is carried out at a temperature of approximately 50°C. In some embodiments, fixing is carried out at ambient temperature.

[0081] In certain embodiments, the method provided herein involves treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 6, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the fixation is carried out at a temperature of approximately 4°C. In one embodiment, the fixation lasts for approximately 1 hour. In one embodiment, the fixation lasts for approximately 1.5 hours. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 2.5 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 3.5 hours. In one embodiment, the fixation lasts for approximately 4 hours.

[0082] In certain embodiments, the method provided herein involves treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 7, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the fixation is carried out at a temperature of approximately 4°C. In one embodiment, the fixation lasts for approximately 1 hour. In one embodiment, the fixation lasts for approximately 1.5 hours. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 2.5 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 3.5 hours. In one embodiment, the fixation lasts for approximately 4 hours.

[0083] In certain embodiments, the method provided herein involves treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 8, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the fixation is carried out at a temperature of approximately 4°C. In one embodiment, the fixation lasts for approximately 1 hour. In one embodiment, the fixation lasts for approximately 1.5 hours. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 2.5 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 3.5 hours. In one embodiment, the fixation lasts for approximately 4 hours.

[0084] In certain embodiments, the method provided herein involves treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 6, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the fixation is carried out at ambient temperature. In one embodiment, the fixation lasts for approximately 1 hour. In one embodiment, the fixation lasts for approximately 1.5 hours. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 2.5 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 3.5 hours. In one embodiment, the fixation lasts for approximately 4 hours.

[0085] In certain embodiments, the method provided herein involves treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 7, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the fixation is carried out at ambient temperature. In one embodiment, the fixation lasts for approximately 1 hour. In one embodiment, the fixation lasts for approximately 1.5 hours. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 2.5 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 3.5 hours. In one embodiment, the fixation lasts for approximately 4 hours.

[0086] In certain embodiments, the method provided herein involves treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 8, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the fixation is carried out at ambient temperature. In one embodiment, the fixation lasts for approximately 1 hour. In one embodiment, the fixation lasts for approximately 1.5 hours. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 2.5 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 3.5 hours. In one embodiment, the fixation lasts for approximately 4 hours.

[0087] In certain embodiments, the method provided herein involves treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 6, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the fixation is carried out at a temperature of approximately 50°C. In one embodiment, the fixation lasts for approximately 1 hour. In one embodiment, the fixation lasts for approximately 1.5 hours. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 2.5 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 3.5 hours. In one embodiment, the fixation lasts for approximately 4 hours.

[0088] In certain embodiments, the method provided herein involves treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 7, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the fixation is carried out at a temperature of approximately 50°C. In one embodiment, the fixation lasts for approximately 1 hour. In one embodiment, the fixation lasts for approximately 1.5 hours. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 2.5 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 3.5 hours. In one embodiment, the fixation lasts for approximately 4 hours.

[0089] In certain embodiments, the method provided herein involves treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 8, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the fixation is carried out at a temperature of approximately 50°C. In one embodiment, the fixation lasts for approximately 1 hour. In one embodiment, the fixation lasts for approximately 1.5 hours. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 2.5 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 3.5 hours. In one embodiment, the fixation lasts for approximately 4 hours.

[0090] In certain embodiments, the method provided herein includes treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer solution with a pH of approximately 6, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the fixation is carried out at a temperature of approximately 4°C. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 4 hours. In one embodiment, the fixation lasts for approximately 5 hours. In one embodiment, the fixation lasts for approximately 6 hours. In one embodiment, the fixation lasts for approximately 7 hours. In one embodiment, the fixation lasts for approximately 8 hours. In one embodiment, the fixation lasts for approximately 9 hours. In one embodiment, the fixation lasts for approximately 10 hours. In one embodiment, the fixation lasts for approximately 11 hours. In one embodiment, the fixation lasts for approximately 12 hours. In one embodiment, the fixation lasts for approximately 13 hours. In one embodiment, the fixation lasts for approximately 14 hours. In one embodiment, the fixation lasts for approximately 15 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 17 hours. In one embodiment, the fixation lasts for approximately 18 hours. In another embodiment, the fixation lasts for approximately 24 hours. In yet another embodiment, the fixation lasts for approximately 36 hours. In yet another embodiment, the fixation lasts for approximately 48 hours.

[0091] In certain embodiments, the method provided herein includes treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer solution with a pH of approximately 7, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the fixation is carried out at a temperature of approximately 4°C. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 4 hours. In one embodiment, the fixation lasts for approximately 5 hours. In one embodiment, the fixation lasts for approximately 6 hours. In one embodiment, the fixation lasts for approximately 7 hours. In one embodiment, the fixation lasts for approximately 8 hours. In one embodiment, the fixation lasts for approximately 9 hours. In one embodiment, the fixation lasts for approximately 10 hours. In one embodiment, the fixation lasts for approximately 11 hours. In one embodiment, the fixation lasts for approximately 12 hours. In one embodiment, the fixation lasts for approximately 13 hours. In one embodiment, the fixation lasts for approximately 14 hours. In one embodiment, the fixation lasts for approximately 15 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 17 hours. In one embodiment, the fixation lasts for approximately 18 hours. In another embodiment, the fixation lasts for approximately 24 hours. In yet another embodiment, the fixation lasts for approximately 36 hours. In yet another embodiment, the fixation lasts for approximately 48 hours.

[0092] In certain embodiments, the method provided herein includes treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer solution with a pH of approximately 8, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the fixation is carried out at a temperature of approximately 4°C. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 4 hours. In one embodiment, the fixation lasts for approximately 5 hours. In one embodiment, the fixation lasts for approximately 6 hours. In one embodiment, the fixation lasts for approximately 7 hours. In one embodiment, the fixation lasts for approximately 8 hours. In one embodiment, the fixation lasts for approximately 9 hours. In one embodiment, the fixation lasts for approximately 10 hours. In one embodiment, the fixation lasts for approximately 11 hours. In one embodiment, the fixation lasts for approximately 12 hours. In one embodiment, the fixation lasts for approximately 13 hours. In one embodiment, the fixation lasts for approximately 14 hours. In one embodiment, the fixation lasts for approximately 15 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 17 hours. In one embodiment, the fixation lasts for approximately 18 hours. In another embodiment, the fixation lasts for approximately 24 hours. In yet another embodiment, the fixation lasts for approximately 36 hours. In yet another embodiment, the fixation lasts for approximately 48 hours.

[0093] In certain embodiments, the method provided herein includes treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer solution with a pH of approximately 6, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the fixation is carried out at ambient temperature. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 4 hours. In one embodiment, the fixation lasts for approximately 5 hours. In one embodiment, the fixation lasts for approximately 6 hours. In one embodiment, the fixation lasts for approximately 7 hours. In one embodiment, the fixation lasts for approximately 8 hours. In one embodiment, the fixation lasts for approximately 9 hours. In one embodiment, the fixation lasts for approximately 10 hours. In one embodiment, the fixation lasts for approximately 11 hours. In one embodiment, the fixation lasts for approximately 12 hours. In one embodiment, the fixation lasts for approximately 13 hours. In one embodiment, the fixation lasts for approximately 14 hours. In one embodiment, the fixation lasts for approximately 15 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 17 hours. In one embodiment, the fixation lasts for approximately 18 hours. In another embodiment, the fixation lasts for approximately 24 hours. In yet another embodiment, the fixation lasts for approximately 36 hours. In yet another embodiment, the fixation lasts for approximately 48 hours.

[0094] In certain embodiments, the method provided herein includes treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer solution with a pH of approximately 7, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the fixation is carried out at ambient temperature. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 4 hours. In one embodiment, the fixation lasts for approximately 5 hours. In one embodiment, the fixation lasts for approximately 6 hours. In one embodiment, the fixation lasts for approximately 7 hours. In one embodiment, the fixation lasts for approximately 8 hours. In one embodiment, the fixation lasts for approximately 9 hours. In one embodiment, the fixation lasts for approximately 10 hours. In one embodiment, the fixation lasts for approximately 11 hours. In one embodiment, the fixation lasts for approximately 12 hours. In one embodiment, the fixation lasts for approximately 13 hours. In one embodiment, the fixation lasts for approximately 14 hours. In one embodiment, the fixation lasts for approximately 15 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 17 hours. In one embodiment, the fixation lasts for approximately 18 hours. In another embodiment, the fixation lasts for approximately 24 hours. In yet another embodiment, the fixation lasts for approximately 36 hours. In yet another embodiment, the fixation lasts for approximately 48 hours.

[0095] In certain embodiments, the method provided herein includes treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer solution with a pH of approximately 8, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the fixation is carried out at ambient temperature. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 4 hours. In one embodiment, the fixation lasts for approximately 5 hours. In one embodiment, the fixation lasts for approximately 6 hours. In one embodiment, the fixation lasts for approximately 7 hours. In one embodiment, the fixation lasts for approximately 8 hours. In one embodiment, the fixation lasts for approximately 9 hours. In one embodiment, the fixation lasts for approximately 10 hours. In one embodiment, the fixation lasts for approximately 11 hours. In one embodiment, the fixation lasts for approximately 12 hours. In one embodiment, the fixation lasts for approximately 13 hours. In one embodiment, the fixation lasts for approximately 14 hours. In one embodiment, the fixation lasts for approximately 15 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 17 hours. In one embodiment, the fixation lasts for approximately 18 hours. In another embodiment, the fixation lasts for approximately 24 hours. In yet another embodiment, the fixation lasts for approximately 36 hours. In yet another embodiment, the fixation lasts for approximately 48 hours.

[0096] In certain embodiments, the method provided herein includes treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer solution with a pH of approximately 6, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the fixation is carried out at a temperature of approximately 50°C. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 4 hours. In one embodiment, the fixation lasts for approximately 5 hours. In one embodiment, the fixation lasts for approximately 6 hours. In one embodiment, the fixation lasts for approximately 7 hours. In one embodiment, the fixation lasts for approximately 8 hours. In one embodiment, the fixation lasts for approximately 9 hours. In one embodiment, the fixation lasts for approximately 10 hours. In one embodiment, the fixation lasts for approximately 11 hours. In one embodiment, the fixation lasts for approximately 12 hours. In one embodiment, the fixation lasts for approximately 13 hours. In one embodiment, the fixation lasts for approximately 14 hours. In one embodiment, the fixation lasts for approximately 15 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 17 hours. In one embodiment, the fixation lasts for approximately 18 hours. In another embodiment, the fixation lasts for approximately 24 hours. In yet another embodiment, the fixation lasts for approximately 36 hours. In yet another embodiment, the fixation lasts for approximately 48 hours.

[0097] In certain embodiments, the method provided herein includes treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer solution with a pH of approximately 7, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the fixation is carried out at a temperature of approximately 50°C. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 4 hours. In one embodiment, the fixation lasts for approximately 5 hours. In one embodiment, the fixation lasts for approximately 6 hours. In one embodiment, the fixation lasts for approximately 7 hours. In one embodiment, the fixation lasts for approximately 8 hours. In one embodiment, the fixation lasts for approximately 9 hours. In one embodiment, the fixation lasts for approximately 10 hours. In one embodiment, the fixation lasts for approximately 11 hours. In one embodiment, the fixation lasts for approximately 12 hours. In one embodiment, the fixation lasts for approximately 13 hours. In one embodiment, the fixation lasts for approximately 14 hours. In one embodiment, the fixation lasts for approximately 15 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 17 hours. In one embodiment, the fixation lasts for approximately 18 hours. In another embodiment, the fixation lasts for approximately 24 hours. In yet another embodiment, the fixation lasts for approximately 36 hours. In yet another embodiment, the fixation lasts for approximately 48 hours.

[0098] In certain embodiments, the method provided herein includes treating a pre-fixed sample with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer solution with a pH of approximately 8, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the fixation is carried out at a temperature of approximately 50°C. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 4 hours. In one embodiment, the fixation lasts for approximately 5 hours. In one embodiment, the fixation lasts for approximately 6 hours. In one embodiment, the fixation lasts for approximately 7 hours. In one embodiment, the fixation lasts for approximately 8 hours. In one embodiment, the fixation lasts for approximately 9 hours. In one embodiment, the fixation lasts for approximately 10 hours. In one embodiment, the fixation lasts for approximately 11 hours. In one embodiment, the fixation lasts for approximately 12 hours. In one embodiment, the fixation lasts for approximately 13 hours. In one embodiment, the fixation lasts for approximately 14 hours. In one embodiment, the fixation lasts for approximately 15 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 16 hours. In one embodiment, the fixation lasts for approximately 17 hours. In one embodiment, the fixation lasts for approximately 18 hours. In another embodiment, the fixation lasts for approximately 24 hours. In yet another embodiment, the fixation lasts for approximately 36 hours. In yet another embodiment, the fixation lasts for approximately 48 hours.

[0099] In certain embodiments, the method provided herein involves treating a pre-fixed sample with formaldehyde equal to or higher than about 12% in an isotonic buffer at a neutral or near-neutral pH, and the fixation is carried out at ambient temperature. In one embodiment, the fixation lasts about 0.5 hours. In one embodiment, the fixation lasts about 1 hour. In one embodiment, the fixation lasts about 1.5 hours. In one embodiment, the fixation lasts about 2 hours. In one embodiment, the fixation lasts about 2.5 hours. In one embodiment, the fixation lasts about 3 hours. In one embodiment, the fixation lasts about 3.5 hours.

[0100] In certain embodiments, the method provided herein involves treating a pre-fixed sample with less than 12% formaldehyde in an isotonic buffer at a neutral or near-neutral pH, and the fixation is carried out at ambient temperature. In one embodiment, the fixation lasts for about 12 hours. In one embodiment, the fixation lasts for about 14 hours. In one embodiment, the fixation lasts for about 16 hours. In one embodiment, the fixation lasts for about 18 hours. In one embodiment, the fixation lasts for about 20 hours. In one embodiment, the fixation lasts for about 22 hours. In one embodiment, the fixation lasts for about 24 hours.

[0101] In certain embodiments, the method provided herein involves treating a pre-fixed sample with about 12% formaldehyde in phosphate-buffered saline, wherein the fixation is carried out at ambient temperature. In one embodiment, the fixation lasts about 1 hour. In one embodiment, the fixation lasts about 1.5 hours. In one embodiment, the fixation lasts about 2 hours. In one embodiment, the fixation lasts about 2.5 hours. In one embodiment, the fixation lasts about 3 hours. In one embodiment, the fixation lasts about 3.5 hours.

[0102] In certain embodiments, the method provided herein involves treating a pre-fixed sample with approximately 24% formaldehyde in phosphate-buffered saline, wherein the fixation is carried out at ambient temperature. In one embodiment, the fixation lasts for approximately 1 hour. In one embodiment, the fixation lasts for approximately 1.5 hours. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 2.5 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 3.5 hours.

[0103] In certain embodiments, the method provided herein involves treating a pre-fixed sample with approximately 37% formaldehyde in phosphate-buffered saline, wherein the fixation is carried out at ambient temperature. In one embodiment, the fixation lasts for approximately 1 hour. In one embodiment, the fixation lasts for approximately 1.5 hours. In one embodiment, the fixation lasts for approximately 2 hours. In one embodiment, the fixation lasts for approximately 2.5 hours. In one embodiment, the fixation lasts for approximately 3 hours. In one embodiment, the fixation lasts for approximately 3.5 hours.

[0104] In certain embodiments, the method provided herein involves treating a pre-fixed sample with about 10% neutral buffered formalin, wherein the fixation is carried out at ambient temperature. In one embodiment, the fixation lasts about 12 hours. In one embodiment, the fixation lasts about 14 hours. In one embodiment, the fixation lasts about 16 hours. In one embodiment, the fixation lasts about 18 hours. In one embodiment, the fixation lasts about 20 hours. In one embodiment, the fixation lasts about 22 hours. In one embodiment, the fixation lasts about 24 hours.

[0105] In other embodiments, this specification provides a method for preparing a biological sample for in situ hybridization, comprising: a first fixation step of fixing the biological sample with a drug; and a post-fixation step of fixing the biological sample with an aldehyde-containing fixative after the first fixation step and before in situ hybridization.

[0106] In some embodiments, the first fixation step is physical fixation. In one embodiment, the first fixation step is heating. In one embodiment, the first fixation step is microwave exposure. In one embodiment, the first fixation step is cryopreservation or freeze-drying. In other embodiments, the first fixation step is chemical fixation using fixative(s). In some embodiments, the first fixation step is chemical fixation by immersion of the biological sample in fixative(s). In some embodiments, the first fixation step is chemical fixation by perfusion of the biological sample with fixative(s). In some embodiments, the first fixation step is chemical fixation by application of vaporized fixative(s).

[0107] In some embodiments, the first fixation step is chemical fixation using a coagulating fixative(s). In other embodiments, the first fixation step is chemical fixation using a non-coagulating fixative(s). In some embodiments, the first fixation step is chemical fixation using a fixative(s) having denaturing properties that remove water from the biological sample. In other embodiments, the first fixation step is chemical fixation using a crosslinking fixative(s) that forms chemical bonds between molecules of the biological sample. In some embodiments, the first fixation step is chemical fixation using an additive fixative(s) (i.e., the fixative(s) becomes part of the biological sample that is processed in subsequent steps of the histological protocol). In other embodiments, the first fixation step is chemical fixation using a non-additive fixative(s) (i.e., the fixative(s) is removed from the biological sample in subsequent steps of the histological protocol once the fixation is performed and completed).

[0108] In one embodiment, the first fixation step is chemical fixation with ethanol. In one embodiment, the first fixation step is chemical fixation with methanol. In one embodiment, the first fixation step is chemical fixation with acetone. In one embodiment, the first fixation step is chemical fixation with acetic acid. In one embodiment, the first fixation step is chemical fixation with zinc chloride. In one embodiment, the first fixation step is chemical fixation with zinc sulfate. In one embodiment, the first fixation step is chemical fixation with picric acid. In one embodiment, the first fixation step is chemical fixation with formaldehyde. In one embodiment, the first fixation step is chemical fixation with glutaraldehyde. In one embodiment, the first fixation step is chemical fixation with osmium tetroxide. In some embodiments, the first fixation step is chemical fixation with carbodiimide. In some embodiments, the first fixation step is chemical fixation with diimide ester. In one embodiment, the first fixation step is chemical fixation with chloro-s-triazide (cyanuryl chloride). In some embodiments, the first fixation step is chemical fixation with diisocyanate. In one embodiment, the first fixation step is chemical fixation with diethyl pyrocarbonate (DPC). In some embodiments, the first fixation step is chemical fixation with maleimide. In one embodiment, the first fixation step is chemical fixation with benzoquinone. In one embodiment, the first fixation step is chemical fixation with mercury chloride. In one embodiment, the first fixation step is chemical fixation with potassium dichromate. In one embodiment, the first fixation step is chemical fixation with potassium permanganate. In one embodiment, the first fixation step is chemical fixation with chromic acid.

[0109] In one embodiment, the first fixation step is chemical fixation with Bouin fixative, which is a solution of picric acid, formaldehyde, and acetic acid. In one embodiment, the first fixation step is chemical fixation with Clarke fixative, which is a solution of ethanol and acetic acid. In one embodiment, the first fixation step is chemical fixation with Carnoy fixative, which is a solution of ethanol, chloroform, and acetic acid. In one embodiment, the first fixation step is chemical fixation with a mixed solution of formaldehyde and glutaraldehyde. In one embodiment, the first fixation step is chemical fixation with FAA, which is a solution of ethanol, acetic acid, and formaldehyde. In one embodiment, the first fixation step is chemical fixation with periodate-lysine-paraformaldehyde (PLP), which is a solution of paraformaldehyde, L-lysine, and INaO4. In one embodiment, the first fixation step is chemical fixation with phosphate-buffered formalin (PBF). In one embodiment, the first fixation step is chemical fixation with Formal Calcium, a solution of formaldehyde and calcium chloride. In one embodiment, the first fixation step is chemical fixation with Formal Physiological Saline, a solution of formaldehyde and sodium chloride. In one embodiment, the first fixation step is chemical fixation with zinc formalin, a solution of formaldehyde and zinc sulfate. In one embodiment, the first fixation step is chemical fixation with Zenker fixative, a solution of mercury chloride, potassium dichromate, and glacial acetic acid. In one embodiment, the first fixation step is chemical fixation with Helly fixative, a solution of formaldehyde, potassium dichromate, sodium sulfate, and mercury chloride. In one embodiment, the first fixation step is chemical fixation with B-5 fixative, c mercury chloride, and sodium acetate. In one embodiment, the first fixation step is chemical fixation using Hollande fixative, which is a solution of formaldehyde, copper acetate, picric acid, and acetic acid. In another embodiment, the first fixation step is chemical fixation using Gendre solution, which is a solution of formaldehyde, ethanol, picric acid, and glacial acetic acid.In one embodiment, the first fixation step is chemical fixation with Methacarn fixative, which is a solution of methanol, chloroform, and glacial acetic acid. In another embodiment, the first fixation step is chemical fixation with alcohol-formalin, which is a solution of formaldehyde, ethanol, and calcium acetate. In yet another embodiment, the first fixation step is chemical fixation with formalic acid alcohol, which is a solution of formaldehyde, glacial acetic acid, and ethanol.

[0110] In some embodiments, the first fixation step is chemical fixation with a mixture solution of two, three, four, or five or more fixatives selected from a list including ethanol, methanol, acetone, acetic acid, zinc chloride, zinc sulfate, picric acid, osmium tetroxide, formaldehyde, glutaraldehyde, carbodiimide, diimide esters, chloro-s-triazide (cyanuric chloride), diisocyanate, diethyl pyrocarbonate (DPC), maleimide, benzoquinone, mercury chloride, potassium dichromate, potassium permanganate, and chromic acid.

[0111] In some embodiments, the first fixation step is chemical fixation with two or more fixatives applied sequentially, though not simultaneously. The two or more fixatives are selected from a list including ethanol, methanol, acetone, acetic acid, zinc chloride, zinc sulfate, picric acid, osmium tetroxide, formaldehyde, glutaraldehyde, carbodiimide, diimide esters, chloro-s-triazide (cyanuric chloride), diisocyanate, diethyl pyrocarbonate (DPC), maleimide, benzoquinone, mercury chloride, potassium dichromate, potassium permanganate, and chromic acid.

[0112] In some embodiments, the first fixation step is chemical fixation with a fixative(s) suitable for preserving nucleic acids. In one embodiment, the first fixation step is chemical fixation with FineFix (see Kothmaier et al., Arch. Pathol. Lab. Med. 135:744-752, 2011). In one embodiment, the first fixation step is chemical fixation with Glyo-fix (see Lykidis et al., Nucleic Acids Res. 35:e85, 2007). In one embodiment, the first fixation step is chemical fixation with Histochoice (see Vince et al., Anal. Cell. Pathol. 15:119-129, 1997). In one embodiment, the first fixation step is chemical fixation with HOPE (see Kothmaier et al., Arch. Pathol. Lab. Med. 135:744-752, 2011). In one embodiment, the first fixation step is chemical fixation using Neo-Fix (see Paavilainen et al., Histochem.Cytochem.:Official J.Histochem.Soc.58:237-246, 2010). In one embodiment, the first fixation step is chemical fixation using the PAXgene Tissue System (see Nietner et al., Int.J.Patel.461:259-269, 2012). In one embodiment, the first fixation step is chemical fixation using RCL2 (see van Essen et al., Clin.Patel.63:1090-1094, 2010). In one embodiment, the first fixation step is chemical fixation using Streck's Tissue Fixative (see Burns et al., Histochem.Cytochem.57:257-264, 2009). In one embodiment, the first fixation step is chemical fixation using UMFIX (see Nadji et al., Appl. Immunohistochem. Mol. Morphol. 13:277-282, 2005). In another embodiment, the first fixation step is chemical fixation using Z7 (see Lykidis et al., Nucleic Acids Res. 35:e85, 2007).In one embodiment, the first fixation step is chemical fixation using ZBF (see Paavilainen et al., Histochem.Cytochem.:Official J.Histochem.Soc.58:237-246, 2010).

[0113] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative(s) after a first fixation step and before in situ hybridization. In one embodiment, the post-fixation step is chemical fixation with formaldehyde. In one embodiment, the post-fixation step is chemical fixation with glutaraldehyde. In one embodiment, the post-fixation step is chemical fixation with Bouin fixative, which is a solution of picric acid, formaldehyde, and acetic acid. In one embodiment, the post-fixation step is chemical fixation with a mixture of formaldehyde and glutaraldehyde. In one embodiment, the post-fixation step is chemical fixation with FAA, which is a solution of ethanol, acetic acid, and formaldehyde. In one embodiment, the post-fixation step is chemical fixation with periodate-lysine-paraformaldehyde (PLP), which is a solution of paraformaldehyde, L-lysine, and INaO4. In one embodiment, the post-fixation step is chemical fixation with phosphate-buffered formalin (PBF). In one embodiment, the post-fixation step is chemical fixation with Formal Calcium, which is a solution of formaldehyde and calcium chloride. In one embodiment, the post-fixation step is chemical fixation with Formal Physiological Saline Solution, which is a solution of formaldehyde and sodium chloride. In one embodiment, the post-fixation step is chemical fixation with zinc formalin, which is a solution of formaldehyde and zinc sulfate. In one embodiment, the post-fixation step is chemical fixation with Helly Fixative, which is a solution of formaldehyde, potassium dichromate, sodium sulfate, and mercury chloride. In one embodiment, the post-fixation step is chemical fixation with Hollande Fixative, which is a solution of formaldehyde, copper acetate, picric acid, and acetic acid. In one embodiment, the post-fixation step is chemical fixation with Gendre Solution, which is a solution of formaldehyde, ethanol, picric acid, and glacial acetic acid. In one embodiment, the post-fixation step is chemical fixation with alcohol-formalin, which is a solution of formaldehyde, ethanol, and calcium acetate. In another embodiment, the post-fixation step is chemical fixation with formalic acid alcohol, which is a solution of formaldehyde, glacial acetic acid, and ethanol.In one embodiment, the post-fixation step is chemical fixation with a mixture of fixatives, wherein at least one of the fixatives in the mixture is formaldehyde or glutaraldehyde. In another embodiment, the post-fixation step is chemical fixation with fixatives that are not used simultaneously but are used sequentially, wherein at least one of the fixatives is formaldehyde or glutaraldehyde.

[0114] In some embodiments, the aldehyde-containing fixative contains formaldehyde. In other embodiments, the aldehyde-containing fixative contains glutaraldehyde.

[0115] In some embodiments, the aldehyde-containing fixative contains about 5% to about 50% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 10% to about 40% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 12% to about 37% formaldehyde.

[0116] In some embodiments, the aldehyde-containing fixative contains about 5% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 6% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 7% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 8% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 9% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 10% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 11% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 12% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 13% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 14% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 15% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 16% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 17% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 18% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 19% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 20% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 30% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 35% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 40% formaldehyde. In some embodiments, the aldehyde-containing fixative contains about 50% formaldehyde.

[0117] In some embodiments, the sample is treated with an aldehyde-containing fixative for approximately 2 to 30 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for approximately 2 to 25 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for approximately 2 to 20 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for approximately 2 to 18 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for approximately 2 to 15 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for approximately 2 to 10 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for approximately 2 to 5 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for approximately 2 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 3 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 4 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 5 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 6 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 7 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 8 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 9 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 10 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 11 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 12 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 13 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 14 hours in the post-fixation step.In some embodiments, the sample is treated with an aldehyde-containing fixative for about 15 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 16 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 17 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 18 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 19 hours in the post-fixation step. In some embodiments, the sample is treated with an aldehyde-containing fixative for about 20 hours in the post-fixation step.

[0118] In some embodiments, the sample is treated with formaldehyde for approximately 2 to 30 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for approximately 2 to 25 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for approximately 2 to 20 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for approximately 2 to 18 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for approximately 2 to 15 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for approximately 2 to 10 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for approximately 2 to 5 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for approximately 2 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for about 3 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for about 4 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for about 5 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for about 6 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for about 7 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for about 8 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for about 9 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for about 10 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for about 11 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for about 12 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for about 13 hours in a post-fixation step. In some embodiments, the sample is treated with formaldehyde for about 14 hours in a post-fixation step. In some embodiments, the sample is treated with formaldehyde for about 15 hours in a post-fixation step.In some embodiments, the sample is treated with formaldehyde for about 16 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for about 17 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for about 18 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for about 19 hours in the post-fixation step. In some embodiments, the sample is treated with formaldehyde for about 20 hours in the post-fixation step.

[0119] In some specific embodiments, the sample is treated in a post-fixation step with an aldehyde-containing fixative containing less than 12% formaldehyde for more than 15 hours. In some specific embodiments, the sample is treated in a post-fixation step with an aldehyde-containing fixative containing less than 12% formaldehyde for about 18 hours. In some specific embodiments, the sample is treated in a post-fixation step with an aldehyde-containing fixative containing less than 12% formaldehyde for about 19 hours. In some specific embodiments, the sample is treated in a post-fixation step with an aldehyde-containing fixative containing less than 12% formaldehyde for about 20 hours. In some specific embodiments, the sample is treated in a post-fixation step with an aldehyde-containing fixative containing less than 12% formaldehyde for about 25 hours.

[0120] In other specific embodiments, the sample is treated in a post-fixation step with an aldehyde-containing fixative containing about 12% or more formaldehyde for less than 10 hours. In other specific embodiments, the sample is treated in a post-fixation step with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 10 hours. In other specific embodiments, the sample is treated in a post-fixation step with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 9 hours. In other specific embodiments, the sample is treated in a post-fixation step with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 8 hours. In other specific embodiments, the sample is treated in a post-fixation step with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 7 hours. In other specific embodiments, the sample is treated in a post-fixation step with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 6 hours. In other specific embodiments, the sample is treated in a post-fixation step with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 5 hours. In other specific embodiments, the sample is treated in a post-fixation step with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 4 hours. In other specific embodiments, the sample is treated in a post-fixation step with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 3 hours. In other specific embodiments, the sample is treated in a post-fixation step with an aldehyde-containing fixative containing about 12% or more formaldehyde for about 2 hours.

[0121] In some specific embodiments, the aldehyde-containing fixative is in a buffer solution with a pH of approximately 6. In some specific embodiments, the aldehyde-containing fixative is in a buffer solution with a pH of approximately 6.5. In some specific embodiments, the aldehyde-containing fixative is in a buffer solution with a pH of approximately 7. In some specific embodiments, the aldehyde-containing fixative is in a buffer solution with a pH of approximately 7.5. In some specific embodiments, the aldehyde-containing fixative is in a buffer solution with a pH of approximately 8. In some specific embodiments, the aldehyde-containing fixative is in a buffer solution with a pH of approximately 8.5. In some specific embodiments, the aldehyde-containing fixative is in a buffer solution with a pH of approximately 9.

[0122] In some embodiments, the post-fixation process is carried out at a temperature of approximately 4 to 50°C. In some embodiments, the post-fixation process is carried out at a temperature of approximately 4°C. In some embodiments, the post-fixation process is carried out at a temperature of approximately 10°C. In some embodiments, the post-fixation process is carried out at a temperature of approximately 20°C. In some embodiments, the post-fixation process is carried out at a temperature of approximately 25°C. In some embodiments, the post-fixation process is carried out at a temperature of approximately 30°C. In some embodiments, the post-fixation process is carried out at a temperature of approximately 40°C. In some embodiments, the post-fixation process is carried out at a temperature of approximately 50°C. In some embodiments, the post-fixation process is carried out at ambient temperature.

[0123] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 6, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the post-fixation step is carried out at a temperature of approximately 4°C. In one embodiment, the post-fixation step lasts for approximately 1 hour. In one embodiment, the post-fixation step lasts for approximately 1.5 hours. In one embodiment, the post-fixation step lasts for approximately 2 hours. In one embodiment, the post-fixation step lasts for approximately 2.5 hours. In one embodiment, the post-fixation step lasts for approximately 3 hours. In one embodiment, the post-fixation step lasts for approximately 3.5 hours. In one embodiment, the post-fixation step lasts for approximately 4 hours.

[0124] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 7, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the post-fixation step is carried out at a temperature of approximately 4°C. In one embodiment, the post-fixation step lasts for approximately 1 hour. In one embodiment, the post-fixation step lasts for approximately 1.5 hours. In one embodiment, the post-fixation step lasts for approximately 2 hours. In one embodiment, the post-fixation step lasts for approximately 2.5 hours. In one embodiment, the post-fixation step lasts for approximately 3 hours. In one embodiment, the post-fixation step lasts for approximately 3.5 hours. In one embodiment, the post-fixation step lasts for approximately 4 hours.

[0125] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 8, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the post-fixation step is carried out at a temperature of approximately 4°C. In one embodiment, the post-fixation step lasts for approximately 1 hour. In one embodiment, the post-fixation step lasts for approximately 1.5 hours. In one embodiment, the post-fixation step lasts for approximately 2 hours. In one embodiment, the post-fixation step lasts for approximately 2.5 hours. In one embodiment, the post-fixation step lasts for approximately 3 hours. In one embodiment, the post-fixation step lasts for approximately 3.5 hours. In one embodiment, the post-fixation step lasts for approximately 4 hours.

[0126] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 6, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the post-fixation step is carried out at ambient temperature. In one embodiment, the post-fixation step lasts approximately 1 hour. In one embodiment, the post-fixation step lasts approximately 1.5 hours. In one embodiment, the post-fixation step lasts approximately 2 hours. In one embodiment, the post-fixation step lasts approximately 2.5 hours. In one embodiment, the post-fixation step lasts approximately 3 hours. In one embodiment, the post-fixation step lasts approximately 3.5 hours. In one embodiment, the post-fixation step lasts approximately 4 hours.

[0127] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 7, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the post-fixation step is carried out at ambient temperature. In one embodiment, the post-fixation step lasts approximately 1 hour. In one embodiment, the post-fixation step lasts approximately 1.5 hours. In one embodiment, the post-fixation step lasts approximately 2 hours. In one embodiment, the post-fixation step lasts approximately 2.5 hours. In one embodiment, the post-fixation step lasts approximately 3 hours. In one embodiment, the post-fixation step lasts approximately 3.5 hours. In one embodiment, the post-fixation step lasts approximately 4 hours.

[0128] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 8, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the post-fixation step is carried out at ambient temperature. In one embodiment, the post-fixation step lasts for approximately 1 hour. In one embodiment, the post-fixation step lasts for approximately 1.5 hours. In one embodiment, the post-fixation step lasts for approximately 2 hours. In one embodiment, the post-fixation step lasts for approximately 2.5 hours. In one embodiment, the post-fixation step lasts for approximately 3 hours. In one embodiment, the post-fixation step lasts for approximately 3.5 hours. In one embodiment, the post-fixation step lasts for approximately 4 hours.

[0129] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 6, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the post-fixation step is carried out at a temperature of approximately 50°C. In one embodiment, the post-fixation step lasts for approximately 1 hour. In one embodiment, the post-fixation step lasts for approximately 1.5 hours. In one embodiment, the post-fixation step lasts for approximately 2 hours. In one embodiment, the post-fixation step lasts for approximately 2.5 hours. In one embodiment, the post-fixation step lasts for approximately 3 hours. In one embodiment, the post-fixation step lasts for approximately 3.5 hours. In one embodiment, the post-fixation step lasts for approximately 4 hours.

[0130] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 7, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the post-fixation step is carried out at a temperature of approximately 50°C. In one embodiment, the post-fixation step lasts for approximately 1 hour. In one embodiment, the post-fixation step lasts for approximately 1.5 hours. In one embodiment, the post-fixation step lasts for approximately 2 hours. In one embodiment, the post-fixation step lasts for approximately 2.5 hours. In one embodiment, the post-fixation step lasts for approximately 3 hours. In one embodiment, the post-fixation step lasts for approximately 3.5 hours. In one embodiment, the post-fixation step lasts for approximately 4 hours.

[0131] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 8, the percentage of aldehyde in the aldehyde-containing fixative is equal to or higher than approximately 12%, and the post-fixation step is carried out at a temperature of approximately 50°C. In one embodiment, the post-fixation step lasts for approximately 1 hour. In one embodiment, the post-fixation step lasts for approximately 1.5 hours. In one embodiment, the post-fixation step lasts for approximately 2 hours. In one embodiment, the post-fixation step lasts for approximately 2.5 hours. In one embodiment, the post-fixation step lasts for approximately 3 hours. In one embodiment, the post-fixation step lasts for approximately 3.5 hours. In one embodiment, the post-fixation step lasts for approximately 4 hours.

[0132] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 6, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the post-fixation step is carried out at a temperature of approximately 4°C. In one embodiment, the post-fixation step lasts approximately 2 hours. In one embodiment, the post-fixation step lasts approximately 3 hours. In one embodiment, the post-fixation step lasts approximately 4 hours. In one embodiment, the post-fixation step lasts approximately 5 hours. In one embodiment, the post-fixation step lasts approximately 6 hours. In one embodiment, the post-fixation step lasts approximately 7 hours. In one embodiment, the post-fixation step lasts approximately 8 hours. In one embodiment, the post-fixation step lasts approximately 9 hours. In one embodiment, the post-fixation step lasts approximately 10 hours. In one embodiment, the post-fixation step lasts approximately 11 hours. In one embodiment, the post-fixation step lasts approximately 12 hours. In one embodiment, the post-fixation step lasts approximately 13 hours. In one embodiment, the post-fixation step lasts approximately 14 hours. In one embodiment, the post-fixing process lasts approximately 15 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 17 hours. In one embodiment, the post-fixing process lasts approximately 18 hours. In one embodiment, the post-fixing process lasts approximately 24 hours. In one embodiment, the post-fixing process lasts approximately 36 hours. In one embodiment, the post-fixing process lasts approximately 48 hours.

[0133] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 7, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the post-fixation step is carried out at a temperature of approximately 4°C. In one embodiment, the post-fixation step lasts approximately 2 hours. In one embodiment, the post-fixation step lasts approximately 3 hours. In one embodiment, the post-fixation step lasts approximately 4 hours. In one embodiment, the post-fixation step lasts approximately 5 hours. In one embodiment, the post-fixation step lasts approximately 6 hours. In one embodiment, the post-fixation step lasts approximately 7 hours. In one embodiment, the post-fixation step lasts approximately 8 hours. In one embodiment, the post-fixation step lasts approximately 9 hours. In one embodiment, the post-fixation step lasts approximately 10 hours. In one embodiment, the post-fixation step lasts approximately 11 hours. In one embodiment, the post-fixation step lasts approximately 12 hours. In one embodiment, the post-fixation step lasts approximately 13 hours. In one embodiment, the post-fixation step lasts approximately 14 hours. In one embodiment, the post-fixing process lasts approximately 15 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 17 hours. In one embodiment, the post-fixing process lasts approximately 18 hours. In one embodiment, the post-fixing process lasts approximately 24 hours. In one embodiment, the post-fixing process lasts approximately 36 hours. In one embodiment, the post-fixing process lasts approximately 48 hours.

[0134] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 8, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the post-fixation step is carried out at a temperature of approximately 4°C. In one embodiment, the post-fixation step lasts approximately 2 hours. In one embodiment, the post-fixation step lasts approximately 3 hours. In one embodiment, the post-fixation step lasts approximately 4 hours. In one embodiment, the post-fixation step lasts approximately 5 hours. In one embodiment, the post-fixation step lasts approximately 6 hours. In one embodiment, the post-fixation step lasts approximately 7 hours. In one embodiment, the post-fixation step lasts approximately 8 hours. In one embodiment, the post-fixation step lasts approximately 9 hours. In one embodiment, the post-fixation step lasts approximately 10 hours. In one embodiment, the post-fixation step lasts approximately 11 hours. In one embodiment, the post-fixation step lasts approximately 12 hours. In one embodiment, the post-fixation step lasts approximately 13 hours. In one embodiment, the post-fixation step lasts approximately 14 hours. In one embodiment, the post-fixing process lasts approximately 15 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 17 hours. In one embodiment, the post-fixing process lasts approximately 18 hours. In one embodiment, the post-fixing process lasts approximately 24 hours. In one embodiment, the post-fixing process lasts approximately 36 hours. In one embodiment, the post-fixing process lasts approximately 48 hours.

[0135] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 6, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the post-fixation step is carried out at ambient temperature. In one embodiment, the post-fixation step lasts approximately 2 hours. In one embodiment, the post-fixation step lasts approximately 3 hours. In one embodiment, the post-fixation step lasts approximately 4 hours. In one embodiment, the post-fixation step lasts approximately 5 hours. In one embodiment, the post-fixation step lasts approximately 6 hours. In one embodiment, the post-fixation step lasts approximately 7 hours. In one embodiment, the post-fixation step lasts approximately 8 hours. In one embodiment, the post-fixation step lasts approximately 9 hours. In one embodiment, the post-fixation step lasts approximately 10 hours. In one embodiment, the post-fixation step lasts approximately 11 hours. In one embodiment, the post-fixation step lasts approximately 12 hours. In one embodiment, the post-fixation step lasts approximately 13 hours. In one embodiment, the post-fixation step lasts approximately 14 hours. In one embodiment, the post-fixing process lasts approximately 15 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 17 hours. In one embodiment, the post-fixing process lasts approximately 18 hours. In one embodiment, the post-fixing process lasts approximately 24 hours. In one embodiment, the post-fixing process lasts approximately 36 hours. In one embodiment, the post-fixing process lasts approximately 48 hours.

[0136] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 7, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the post-fixation step is carried out at ambient temperature. In one embodiment, the post-fixation step lasts approximately 2 hours. In one embodiment, the post-fixation step lasts approximately 3 hours. In one embodiment, the post-fixation step lasts approximately 4 hours. In one embodiment, the post-fixation step lasts approximately 5 hours. In one embodiment, the post-fixation step lasts approximately 6 hours. In one embodiment, the post-fixation step lasts approximately 7 hours. In one embodiment, the post-fixation step lasts approximately 8 hours. In one embodiment, the post-fixation step lasts approximately 9 hours. In one embodiment, the post-fixation step lasts approximately 10 hours. In one embodiment, the post-fixation step lasts approximately 11 hours. In one embodiment, the post-fixation step lasts approximately 12 hours. In one embodiment, the post-fixation step lasts approximately 13 hours. In one embodiment, the post-fixation step lasts approximately 14 hours. In one embodiment, the post-fixing process lasts approximately 15 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 17 hours. In one embodiment, the post-fixing process lasts approximately 18 hours. In one embodiment, the post-fixing process lasts approximately 24 hours. In one embodiment, the post-fixing process lasts approximately 36 hours. In one embodiment, the post-fixing process lasts approximately 48 hours.

[0137] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 8, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the post-fixation step is carried out at ambient temperature. In one embodiment, the post-fixation step lasts approximately 2 hours. In one embodiment, the post-fixation step lasts approximately 3 hours. In one embodiment, the post-fixation step lasts approximately 4 hours. In one embodiment, the post-fixation step lasts approximately 5 hours. In one embodiment, the post-fixation step lasts approximately 6 hours. In one embodiment, the post-fixation step lasts approximately 7 hours. In one embodiment, the post-fixation step lasts approximately 8 hours. In one embodiment, the post-fixation step lasts approximately 9 hours. In one embodiment, the post-fixation step lasts approximately 10 hours. In one embodiment, the post-fixation step lasts approximately 11 hours. In one embodiment, the post-fixation step lasts approximately 12 hours. In one embodiment, the post-fixation step lasts approximately 13 hours. In one embodiment, the post-fixation step lasts approximately 14 hours. In one embodiment, the post-fixing process lasts approximately 15 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 17 hours. In one embodiment, the post-fixing process lasts approximately 18 hours. In one embodiment, the post-fixing process lasts approximately 24 hours. In one embodiment, the post-fixing process lasts approximately 36 hours. In one embodiment, the post-fixing process lasts approximately 48 hours.

[0138] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 6, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the post-fixation step is carried out at a temperature of approximately 50°C. In one embodiment, the post-fixation step lasts for approximately 2 hours. In one embodiment, the post-fixation step lasts for approximately 3 hours. In one embodiment, the post-fixation step lasts for approximately 4 hours. In one embodiment, the post-fixation step lasts for approximately 5 hours. In one embodiment, the post-fixation step lasts for approximately 6 hours. In one embodiment, the post-fixation step lasts for approximately 7 hours. In one embodiment, the post-fixation step lasts for approximately 8 hours. In one embodiment, the post-fixation step lasts for approximately 9 hours. In one embodiment, the post-fixation step lasts for approximately 10 hours. In one embodiment, the post-fixation step lasts for approximately 11 hours. In one embodiment, the post-fixation step lasts for approximately 12 hours. In one embodiment, the post-fixation step lasts for approximately 13 hours. In one embodiment, the post-fixation step lasts for approximately 14 hours. In one embodiment, the post-fixing process lasts approximately 15 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 17 hours. In one embodiment, the post-fixing process lasts approximately 18 hours. In one embodiment, the post-fixing process lasts approximately 24 hours. In one embodiment, the post-fixing process lasts approximately 36 hours. In one embodiment, the post-fixing process lasts approximately 48 hours.

[0139] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 7, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the post-fixation step is carried out at a temperature of approximately 50°C. In one embodiment, the post-fixation step lasts for approximately 2 hours. In one embodiment, the post-fixation step lasts for approximately 3 hours. In one embodiment, the post-fixation step lasts for approximately 4 hours. In one embodiment, the post-fixation step lasts for approximately 5 hours. In one embodiment, the post-fixation step lasts for approximately 6 hours. In one embodiment, the post-fixation step lasts for approximately 7 hours. In one embodiment, the post-fixation step lasts for approximately 8 hours. In one embodiment, the post-fixation step lasts for approximately 9 hours. In one embodiment, the post-fixation step lasts for approximately 10 hours. In one embodiment, the post-fixation step lasts for approximately 11 hours. In one embodiment, the post-fixation step lasts for approximately 12 hours. In one embodiment, the post-fixation step lasts for approximately 13 hours. In one embodiment, the post-fixation step lasts for approximately 14 hours. In one embodiment, the post-fixing process lasts approximately 15 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 17 hours. In one embodiment, the post-fixing process lasts approximately 18 hours. In one embodiment, the post-fixing process lasts approximately 24 hours. In one embodiment, the post-fixing process lasts approximately 36 hours. In one embodiment, the post-fixing process lasts approximately 48 hours.

[0140] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with an aldehyde-containing fixative, wherein the aldehyde-containing fixative is in an isotonic buffer at pH approximately 8, the percentage of aldehyde in the aldehyde-containing fixative is less than approximately 12%, and the post-fixation step is carried out at a temperature of approximately 50°C. In one embodiment, the post-fixation step lasts for approximately 2 hours. In one embodiment, the post-fixation step lasts for approximately 3 hours. In one embodiment, the post-fixation step lasts for approximately 4 hours. In one embodiment, the post-fixation step lasts for approximately 5 hours. In one embodiment, the post-fixation step lasts for approximately 6 hours. In one embodiment, the post-fixation step lasts for approximately 7 hours. In one embodiment, the post-fixation step lasts for approximately 8 hours. In one embodiment, the post-fixation step lasts for approximately 9 hours. In one embodiment, the post-fixation step lasts for approximately 10 hours. In one embodiment, the post-fixation step lasts for approximately 11 hours. In one embodiment, the post-fixation step lasts for approximately 12 hours. In one embodiment, the post-fixation step lasts for approximately 13 hours. In one embodiment, the post-fixation step lasts for approximately 14 hours. In one embodiment, the post-fixing process lasts approximately 15 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 16 hours. In one embodiment, the post-fixing process lasts approximately 17 hours. In one embodiment, the post-fixing process lasts approximately 18 hours. In one embodiment, the post-fixing process lasts approximately 24 hours. In one embodiment, the post-fixing process lasts approximately 36 hours. In one embodiment, the post-fixing process lasts approximately 48 hours.

[0141] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with approximately 12% or higher formaldehyde in an isotonic buffer at a neutral or near-neutral pH, the post-fixation step being carried out at ambient temperature. In one embodiment, the post-fixation step lasts approximately 0.5 hours. In one embodiment, the post-fixation step lasts approximately 1 hour. In one embodiment, the post-fixation step lasts approximately 1.5 hours. In one embodiment, the post-fixation step lasts approximately 2 hours. In one embodiment, the post-fixation step lasts approximately 2.5 hours. In one embodiment, the post-fixation step lasts approximately 3 hours. In one embodiment, the post-fixation step lasts approximately 3.5 hours.

[0142] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with less than 12% formaldehyde in an isotonic buffer at a neutral or near-neutral pH, and the post-fixation step is carried out at ambient temperature. In one embodiment, the post-fixation step lasts for about 12 hours. In one embodiment, the post-fixation step lasts for about 14 hours. In one embodiment, the post-fixation step lasts for about 16 hours. In one embodiment, the post-fixation step lasts for about 18 hours. In one embodiment, the post-fixation step lasts for about 20 hours. In one embodiment, the post-fixation step lasts for about 22 hours. In one embodiment, the post-fixation step lasts for about 24 hours.

[0143] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with approximately 12% formaldehyde in phosphate-buffered saline, the post-fixation step being carried out at ambient temperature. In one embodiment, the post-fixation step lasts for approximately 1 hour. In one embodiment, the post-fixation step lasts for approximately 1.5 hours. In one embodiment, the post-fixation step lasts for approximately 2 hours. In one embodiment, the post-fixation step lasts for approximately 2.5 hours. In one embodiment, the post-fixation step lasts for approximately 3 hours. In one embodiment, the post-fixation step lasts for approximately 3.5 hours.

[0144] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with approximately 24% formaldehyde in phosphate-buffered saline, the post-fixation step being carried out at ambient temperature. In one embodiment, the post-fixation step lasts for approximately 1 hour. In one embodiment, the post-fixation step lasts for approximately 1.5 hours. In one embodiment, the post-fixation step lasts for approximately 2 hours. In one embodiment, the post-fixation step lasts for approximately 2.5 hours. In one embodiment, the post-fixation step lasts for approximately 3 hours. In one embodiment, the post-fixation step lasts for approximately 3.5 hours.

[0145] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with approximately 37% formaldehyde in phosphate-buffered saline, the post-fixation step being carried out at ambient temperature. In one embodiment, the post-fixation step lasts for approximately 1 hour. In one embodiment, the post-fixation step lasts for approximately 1.5 hours. In one embodiment, the post-fixation step lasts for approximately 2 hours. In one embodiment, the post-fixation step lasts for approximately 2.5 hours. In one embodiment, the post-fixation step lasts for approximately 3 hours. In one embodiment, the post-fixation step lasts for approximately 3.5 hours.

[0146] In certain embodiments, the method for preparing a biological sample for in situ hybridization provided herein includes a post-fixation step with 10% neutral buffered formalin, the post-fixation step being carried out at ambient temperature. In one embodiment, the post-fixation step lasts approximately 12 hours. In one embodiment, the post-fixation step lasts approximately 14 hours. In one embodiment, the post-fixation step lasts approximately 16 hours. In one embodiment, the post-fixation step lasts approximately 18 hours. In one embodiment, the post-fixation step lasts approximately 20 hours. In one embodiment, the post-fixation step lasts approximately 22 hours. In one embodiment, the post-fixation step lasts approximately 24 hours.

[0147] In some embodiments of the various methods provided herein, the sample was prepared for in situ hybridization. In some embodiments, in situ hybridization is for detecting a target nucleic acid containing fewer than 100 nucleotides. In some embodiments, the target nucleic acid contains 15 to 100 nucleotides. In some embodiments, the target nucleic acid contains 15 to 80 nucleotides. In some embodiments, the target nucleic acid contains 15 to 60 nucleotides. In some embodiments, the target nucleic acid contains 15 to 50 nucleotides. In some embodiments, the target nucleic acid contains 15 to 40 nucleotides. In some embodiments, the target nucleic acid contains fewer than 90 nucleotides. In some embodiments, the target nucleic acid contains fewer than 80 nucleotides. In some embodiments, the target nucleic acid contains fewer than 70 nucleotides. In some embodiments, the target nucleic acid contains fewer than 60 nucleotides. In some embodiments, the target nucleic acid contains fewer than 50 nucleotides. In some embodiments, the target nucleic acid contains fewer than 40 nucleotides. In some embodiments, the target nucleic acid contains fewer than 30 nucleotides. In some embodiments, the target nucleic acid contains fewer than 20 nucleotides. In some embodiments, the target nucleic acid contains fewer than 16 nucleotides. In some embodiments, in situ hybridization is for detecting DNA. In some embodiments, in situ hybridization is for detecting RNA. The methods provided herein can also be used to detect longer nucleic acids, such as those containing 100, 200, 300, 500, or 1,000 or more nucleotides.

[0148] In some embodiments, the method for preparing biological samples provided herein is for RNA in situ hybridization. In some embodiments, the method for preparing biological samples for in situ hybridization is for detecting low-molecular-weight RNA species. In one embodiment, the number of nucleotides in the RNA to be detected is less than 100. In one embodiment, the number of nucleotides in the RNA to be detected is less than 50. In one embodiment, the number of nucleotides in the RNA to be detected is less than 40. In one embodiment, the number of nucleotides in the RNA to be detected is between 10 and 40. In one embodiment, the number of nucleotides in the RNA to be detected is between 15 and 40. In one embodiment, the number of nucleotides in the RNA to be detected is between 30 and 40.

[0149] In one embodiment, the method is for detecting sncRNA. In one embodiment, the method is for detecting miRNA. In one embodiment, the method is for detecting siRNA. In one embodiment, the method is for detecting piRNA. In one embodiment, the method is for detecting endogenous RNA. In another embodiment, the method is for detecting exogenous RNA. SncRNAs have been shown to have the ability to efficiently regulate gene expression in clinically relevant model systems as beneficial therapeutic agents for disease treatment (Watts et al., Journal of Pathology, 226(2), 365-379, 2012; Schoch et al., Neuron Review, 94, 1056-1070, 2017). miRNAs are naturally occurring, small (approximately 22 nucleotides) regulatory RNAs found in all multicellular organisms, unicellular algae, and some viruses (Molnar et al., Nature, 447(7148), 2007; Bartel, Cell, 173, 20-51, 2018). To date, more than 15,000 miRNAs from animals, plants, and viruses have been registered (www.mirbase.org), many of which are expressed in a manner specific to tissue, cell type, and cell state. Dysregulation of miRNA expression can lead to serious conditions, such as neurological disorders, infertility, immunodeficiency, or cancer.

[0150] In some embodiments, the methods provided herein are for detecting naturally occurring small nucleic acids. Naturally occurring small nucleic acids are responsible for a wide variety of critical cellular functions, from transcription and RNA processing to translation. A common characteristic of these RNAs is their size, with a nucleotide length ranging from 15 to 40. In other embodiments, the methods provided herein are for detecting synthetic small nucleic acids.

[0151] In some embodiments, the methods for preparing biological samples for in situ hybridization provided herein include 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.

[0152] 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, tissues, blood, and cytological samples can be obtained and processed using methods 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.

[0153] Those skilled in the art will understand that any number of suitable cells can be used when detecting target nucleic acids 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 live specimen, autopsy specimen, or forensic material). Biological samples also include samples obtained from areas of living subjects that contain, or are suspected of containing, 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, cell cultures (including primary cell cultures), cell lines, tissues, organelles, organelles, and 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.

[0154] 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).

[0155] 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.) 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).

[0156] In certain embodiments, the sample is a tissue specimen or derived from a tissue specimen. In some embodiments, the tissue specimen is formalin-fixed and paraffin-embedded (FFPE). In some embodiments, the tissue specimen is fresh-frozen. In some embodiments, the tissue specimen is prepared with a fixative other than formalin. In some embodiments, the fixative other than formalin is selected from the group consisting of ethanol, methanol, Bouin fixative, B5, and IBF. In other specific embodiments, the sample is a blood specimen or derived from a blood specimen. In yet another specific embodiment, the sample is a cytological specimen or derived from a cytological specimen.

[0157] 5.3 Method for detecting target nucleic acids In another embodiment, a method for detecting a target nucleic acid in a cell is provided herein, comprising: preparing a biological sample according to a method provided herein, for example, as described in Section 5.2 above; and performing an in situ hybridization detection assay using a set of one or more probes that hybridize to the target nucleic acid.

[0158] In some embodiments, the method provided herein for in situ detection of a target nucleic acid in a pre-fixed sample comprises applying an aldehyde-containing fixative to the sample and performing an in situ hybridization detection assay using a set of one or more probes that hybridize to the target nucleic acid.

[0159] In some embodiments, in situ hybridization is used to detect DNA. In some embodiments, in situ hybridization is used to detect RNA.

[0160] In some embodiments, the methods provided herein detect relatively short nucleic acids. For example, in some embodiments, in situ hybridization is for detecting target nucleic acids containing fewer than 100 nucleotides. In some embodiments, the target nucleic acid contains 15 to 100 nucleotides. In some embodiments, the target nucleic acid contains 15 to 80 nucleotides. In some embodiments, the target nucleic acid contains 15 to 60 nucleotides. In some embodiments, the target nucleic acid contains 15 to 50 nucleotides. In some embodiments, the target nucleic acid contains 15 to 40 nucleotides. In some embodiments, the target nucleic acid contains fewer than 90 nucleotides. In some embodiments, the target nucleic acid contains fewer than 80 nucleotides. In some embodiments, the target nucleic acid contains fewer than 70 nucleotides. In some embodiments, the target nucleic acid contains fewer than 60 nucleotides. In some embodiments, the target nucleic acid contains fewer than 50 nucleotides. In some embodiments, the target nucleic acid contains fewer than 40 nucleotides. In some embodiments, the target nucleic acid contains fewer than 30 nucleotides. In some embodiments, the target nucleic acid contains fewer than 20 nucleotides. In some embodiments, the target nucleic acid contains fewer than 16 nucleotides. In some embodiments, in situ hybridization is for detecting small RNA species. In one embodiment, the number of nucleotides in the RNA to be detected is less than 100. In one embodiment, the number of nucleotides in the RNA to be detected is less than 50. In one embodiment, the number of nucleotides in the RNA to be detected is less than 40. In one embodiment, the number of nucleotides in the RNA to be detected is between 10 and 40. In one embodiment, the number of nucleotides in the RNA to be detected is between 15 and 40. In one embodiment, the number of nucleotides in the RNA to be detected is between 30 and 40. In one embodiment, this method is for detecting sncRNA. In one embodiment, this method is for detecting miRNA. In one embodiment, this method is for detecting siRNA. In one embodiment, this method is for detecting piRNA. In one embodiment, this method is for detecting ASO.In one embodiment, this method is for detecting endogenous RNA. In another embodiment, this method is for detecting exogenous RNA.

[0161] Furthermore, the methods provided herein can also be used to detect relatively long nucleic acids, such as those containing 100, 200, 300, 500, or 1,000 or more nucleotides.

[0162] 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” refers to a type of hybridization in which a directly or indirectly labeled complementary DNA or RNA strand (such as a probe) is used to bind to a given nucleic acid in a sample, specifically a portion or section of tissue or cell (in situ), to locate that nucleic acid. The type of probe may be double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded complementary RNA (sscRNA), messenger RNA (mRNA), microRNA (miRNA), ribosomal RNA, mitochondrial RNA, and / or synthetic oligonucleotides.

[0163] In some embodiments, the in situ hybridization 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.

[0164] 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.

[0165] 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.

[0166] In some specific embodiments, the RNA ISH used herein is RNAscope®, as described in detail, for example, U.S. Patents 7,709,198, 8,604,182, and 8,951,726. Specifically, RNAscope® is described as using a specially designed oligonucleotide probe in combination with 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)).

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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 a polynucleotide sequence in an amplifier, pre-amplifier, or pre-pre-amplifier in an SGC.

[0172] In some embodiments, the SGCs provided herein include additional descriptions with respect to such amplifiers, pre-amplifiers, and / or pre-pre-amplifiers.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] Labels are typically used in in situ hybridization of RNA to detect target nucleic acids. As used herein, “label” refers to the portion of a molecule that facilitates detection. 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.

[0177] 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.

[0178] 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 described, for example, in U.S. Publication No. 2012 / 0100540. A variety of detectable enzyme substrates, including chromogenic 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, substrates are converted into products that form precipitates deposited 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®, Warp Red®, and Biocare's Vulcan Fast Red and Ferangi Blue (Concord CA; biocare.net / products / detection / chromogens).

[0179] Examples of rare earth metals and metal isotopes suitable as detectable labels include, but are not limited to, lanthanide(III) isotopes such as 141Pr, 142Nd, 143Nd, 144Nd, 145Nd, 146Nd, 147Sm, 148Nd, 149Sm, 150Nd, 151Eu, 152Sm, 153Eu, 154Sm, 155Gd, 156Gd, 158Gd, 159Tb, 160Gd, 161Dy, 162Dy, 163Dy, 164Dy, 165Ho, 166Er, 167Er, 168Er, 169Tm, 170Er, 171Yb, 172Yb, 173Yb, 174Yb, 175Lu, and 176Yb. Metallic isotopes can be detected, for example, using time-of-flight mass spectrometry (TOF-MS) (e.g., Fluidigm's Helios and Hyperion systems, fluidigm.com / systems; South San Francisco, CA).

[0180] 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.

[0181] 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).

[0182] 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)).

[0183] 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.

[0184] 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, Burlinghame CA). Various cleavable regions can be included in the linker to allow the label to be cleaved from the label probe. Such cleavable regions include groups that can be cleaved chemically, photochemically, or enzymatically. Cleavable chemical linkers can include cleavable chemical regions 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 can include a site for enzymatic cleavage. For example, the linker can include a protein cleavage site. Generally, such cleavage sites are for sequence-specific proteases. Such proteases include human rhinovirus 3C protease (cleavage site: LEVLFQ / GP), enterokinase (cleavage site: DDDDK / ), and X aExamples of cleavable factors include (cleavage site: IEGR / ), tobacco etch virus protease (cleavage site: ENLYFQ / G), and thrombin (cleavage site: LVPR / GS) (see, e.g., Oxford Genetics, Oxford, UK). Other cleavable regions may be, for example, 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)).

[0185] 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.

[0186] In some embodiments, the methods for detecting target nucleic acids in cells provided herein include a pretreatment step before hybridization of the target probe(s). In some embodiments, the pretreatment step includes a blocking step in which a specific blocking agent(s) is applied to block specific endogenous components of the 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. 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 step of pretreatment immediately after deparaffinization. In some embodiments, the pretreatment step includes an epitope antigen retrieval step in which a specific epitope antigen retrieval buffer(s) can 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 50°C to 100°C. In one embodiment, the epitope antigen retrieval step includes heating the sample to approximately 88°C. In some embodiments, the pretreatment step includes a permeabilization step that retains nucleic acid targets in the cells and allows target probes, signaling complexes, etc., to enter the cells. In some embodiments, the permeabilization step includes digestion with a protease. Surfactants (e.g., Triton X-100 or SDS) and Proteinase K can also 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 nonspecific protease that is active over a wide pH range and is not easily inactivated. Proteinase K is used to digest proteins surrounding target mRNA. The optimal concentration and duration of treatment can be determined experimentally, as is well known in the art. Subsequently, a cell washing step can be performed to remove lysates generated in any step of the pretreatment.In some embodiments, the sample is in formalin-fixed paraffin-embedded tissue, and a deparaffinization step is required when removing the paraffin.

[0187] In some embodiments, the methods for detecting target nucleic acids in cells provided herein include a post-fixation step at a specific timing. In one embodiment, the post-fixation step is (i) after the first fixation step and (ii) before applying at least one set of one or more target probes capable of hybridizing to the target nucleic acid. In one embodiment, the post-fixation step is (i) after the first fixation step and (ii) before the pretreatment step described in the preceding paragraph. In one embodiment, the post-fixation step is (i) after the first fixation step and (ii) before the blocking step described in the preceding paragraph. In one embodiment, the post-fixation step is (i) after the first fixation step and (ii) before the epitope antigen retrieval step described in the preceding paragraph. In one embodiment, the post-fixation step is (i) after the first fixation step and (ii) before the permeabilization step described in the preceding paragraph. In one embodiment, the post-fixation step is (i) after the deparaffinization step and (ii) before applying at least one set of one or more target probes capable of hybridizing to the target nucleic acid. In one embodiment, the post-fixation step is (i) after the deparaffinization step and (ii) before the pretreatment step described in the preceding paragraph. In one embodiment, the post-fixation step is (i) after the deparaffinization step and (ii) before the blocking step described in the preceding paragraph. In one embodiment, the post-fixation step is (i) after the deparaffinization step and (ii) before the epitope antigen retrieval step described in the preceding paragraph. In one embodiment, the post-fixation step is (i) after the deparaffinization step and (ii) before the permeabilization step described in the preceding paragraph.

[0188] The methods provided herein have several applications in research and diagnostics (Hanna et al., Frontiers in Genetics, 10, 1-6, 2019; Watts et al., Journal of Pathology, 226(2), 365-379, 2012). The methods provided herein may improve our understanding of small nucleic acids such as sncRNA, miRNA, siRNA, piRNA, and ASO in their natural context, as well as the gene regulatory networks to which these small nucleic acids are involved in various health and disease processes.

[0189] In some embodiments, the methods provided herein can detect low-molecular-weight RNA with temporal and spatial resolution. In one embodiment, the methods provided herein can be used for tissue and cell type identification. In one embodiment, the methods provided herein can be used for identification of different stages of development. In one embodiment, the methods provided herein can be used for characterizing adult tissue.

[0190] In some embodiments, the methods provided herein can be used to detect the expression of denatured small RNA or the presence of small RNA derived from a pathogen. In one embodiment, the methods provided herein can be used to diagnose a disease or disorder. In one embodiment, the methods provided herein can be used to diagnose a pathogen.

[0191] In some embodiments, the methods provided herein are for monitoring the effectiveness of small RNA-based therapies. In one embodiment, the methods provided herein are for monitoring the effectiveness of siRNA-based therapies. In one embodiment, the methods provided herein are for monitoring the effectiveness of ASO-based therapies. In some embodiments, the methods provided herein are for determining the effectiveness of small RNA-based therapies. In one embodiment, the methods provided herein are for determining the effectiveness of siRNA-based therapies. In one embodiment, the methods provided herein are for determining the effectiveness of ASO-based therapies.

[0192] In certain embodiments, this method can be used to detect the presence of siRNA following delivery of siRNA to a disease model. In certain embodiments, this method can be used to localize siRNA following delivery of siRNA to a disease model. In certain embodiments, this method can be used to quantify siRNA following delivery of siRNA to a disease model. In certain embodiments, this method can be used to quantify the RNA targeted by siRNA following delivery of siRNA to a disease model.

[0193] In certain embodiments, this method can be used to detect the presence of ASO following delivery of ASO to a disease model. In certain embodiments, this method can be used to localize ASO following delivery of ASO to a disease model. In certain embodiments, this method can be used to quantify ASO following delivery of ASO to a disease model. In certain embodiments, this method can be used to quantify the RNA targeted by ASO following delivery of ASO to a disease model.

[0194] 5.4 Kits for in situ detection of target nucleic acids In another embodiment, a kit for carrying out the various methods described herein is provided.

[0195] In some embodiments, the Specified Provisions provide a kit for in situ detection of a target nucleic acid in cells, comprising: (i) an agent for fixing a biological sample containing cells; (ii) an aldehyde-containing fixative for fixing the biological sample; (iii) an agent for performing in situ hybridization; and (iv) instructions indicating that the aldehyde-containing fixative is used after the agent of element (i).

[0196] In other embodiments, the Specified Provision provides a kit for in situ detection of a target nucleic acid in cells, comprising: (i) an aldehyde-containing fixative for fixing a pre-immobilized biological sample containing cells; and (ii) a drug for performing in situ hybridization.

[0197] In one embodiment, the agent used to fix the biological sample in the kit is ethanol. In one embodiment, the agent used to fix the biological sample in the kit is methanol. In one embodiment, the agent used to fix the biological sample in the kit is acetone. In one embodiment, the agent used to fix the biological sample in the kit is acetic acid. In one embodiment, the agent used to fix the biological sample in the kit is zinc chloride. In one embodiment, the agent used to fix the biological sample in the kit is zinc sulfide. In one embodiment, the agent used to fix the biological sample in the kit is picric acid. In one embodiment, the agent used to fix the biological sample in the kit is formaldehyde. In one embodiment, the agent used to fix the biological sample in the kit is glutaraldehyde. In one embodiment, the agent used to fix the biological sample in the kit is osmium tetroxide. In some specific embodiments, the agent used to fix the biological sample in the kit is a type of carbodiimide. In some specific embodiments, the agent used to fix the biological sample in the kit is a type of diimide ester. In one embodiment, the agent used to fix the biological sample in the kit is chloro-s-triazide (cyanuryl chloride). In some specific embodiments, the agent used to fix the biological sample in the kit is a type of diisocyanate. In one embodiment, the agent used to fix the biological sample in the kit is diethyl pyrocarbonate (DPC). In some specific embodiments, the agent used to fix the biological sample in the kit is a type of maleimide. In one embodiment, the agent used to fix the biological sample in the kit is benzoquinone. In one embodiment, the agent used to fix the biological sample in the kit is mercury chloride. In one embodiment, the agent used to fix the biological sample in the kit is potassium dichromate. In one embodiment, the agent used to fix the biological sample in the kit is potassium permanganate. In one embodiment, the agent used to fix the biological sample in the kit is chromic acid.

[0198] In one embodiment, the fixative used to fix the biological sample in the kit is Bouin fixative, which is a solution of picric acid, formaldehyde, and acetic acid. In one embodiment, the fixative used to fix the biological sample in the kit is Clarke fixative, which is a solution of ethanol and acetic acid. In one embodiment, the fixative used to fix the biological sample in the kit is Carnoy fixative, which is a solution of ethanol, chloroform, and acetic acid. In one embodiment, the fixative used to fix the biological sample in the kit is a mixed solution of formaldehyde and glutaraldehyde. In one embodiment, the fixative used to fix the biological sample in the kit is FAA, which is a solution of ethanol, acetic acid, and formaldehyde. In one embodiment, the fixative used to fix the biological sample in the kit is periodate-lysine-paraformaldehyde (PLP), which is a solution of paraformaldehyde, L-lysine, and INaO4. In one embodiment, the fixative used to fix the biological sample in the kit is phosphate-buffered formalin (PBF). In one embodiment, the fixative used to fix the biological sample in the kit is formal calcium, which is a solution of formaldehyde and calcium chloride. In one embodiment, the fixative used to fix the biological sample in the kit is formal saline solution, which is a solution of formaldehyde and sodium chloride. In one embodiment, the fixative used to fix the biological sample in the kit is zinc formalin, which is a solution of formaldehyde and zinc sulfate. In one embodiment, the fixative used to fix the biological sample in the kit is Zenker fixative, which is a solution of mercury chloride, potassium dichromate, and glacial acetic acid. In one embodiment, the fixative used to fix the biological sample in the kit is Helly fixative, which is a solution of formaldehyde, potassium dichromate, sodium sulfate, and mercury chloride. In one embodiment, the fixative used to fix the biological sample in the kit is B-5 fixative, c mercuric chloride, and sodium acetate. In one embodiment, the agent used to fix the biological sample in the kit is Hollande fixative, which is a solution of formaldehyde, copper acetate, picric acid, and acetic acid.In one embodiment, the fixative used to fix the biological sample in the kit is Gendre's solution, which is a solution of formaldehyde, ethanol, picric acid, and glacial acetic acid. In one embodiment, the fixative used to fix the biological sample in the kit is Methacarn fixative, which is a solution of methanol, chloroform, and glacial acetic acid. In one embodiment, the fixative used to fix the biological sample in the kit is alcohol formalin, which is a solution of formaldehyde, ethanol, and calcium acetate. In one embodiment, the fixative used to fix the biological sample in the kit is formalic acid alcohol, which is a solution of formaldehyde, glacial acetic acid, and ethanol.

[0199] In some embodiments, the agents used to fix the biological sample in the kit are a mixture of two, three, four, or five or more fixatives selected from a list including ethanol, methanol, acetone, acetic acid, zinc chloride, zinc sulfate, picric acid, osmium tetroxide, formaldehyde, glutaraldehyde, carbodiimide, diimide esters, chloro-s-triazide (cyanuric chloride), diisocyanate, diethyl pyrocarbonate (DPC), maleimide, benzoquinone, mercury chloride, potassium dichromate, potassium permanganate, and chromic acid.

[0200] In some embodiments, the agents used to fix the biological sample in the kit are two or more fixatives applied sequentially, not simultaneously, and the two or more fixatives are selected from a list including ethanol, methanol, acetone, acetic acid, zinc chloride, zinc sulfate, picric acid, osmium tetroxide, formaldehyde, glutaraldehyde, carbodiimide, diimide esters, chloro-s-triazide (cyanuric chloride), diisocyanate, diethyl pyrocarbonate (DPC), maleimide, benzoquinone, mercury chloride, potassium dichromate, potassium permanganate, and chromic acid.

[0201] In some embodiments, the agent used to fix the biological sample in the kit is a fixative(s) suitable for preserving nucleic acids. In one embodiment, the fixative is FineFix (see Kothmaier et al., Arch. Pathol. Lab. Med. 135:744-752, 2011). In one embodiment, the fixative is Glyo-fix (see Lykidis et al., Nucleic Acids Res. 35:e85, 2007). In one embodiment, the fixative is Histochoice (see Vince et al., Anal. Cell. Pathol. 15:119-129, 1997). In one embodiment, the fixative is HOPE (see Kothmaier et al., Arch. Pathol. Lab. Med. 135:744-752, 2011). In one embodiment, the fixative is Neo-Fix (see Paavilainen et al., Histochem.Cytochem.:Official J.Histochem.Soc.58:237-246, 2010). In one embodiment, the fixative is PAXgene Tissue System (see Nietner et al., Int.J.Patrol.461:259-269, 2012). In one embodiment, the fixative is RCL2 (see van Essen et al., Clin.Patrol.63:1090-1094, 2010). In one embodiment, the fixative is Streck's Tissue Fixative (see Burns et al., Histochem.Cytochem.57:257-264, 2009). In one embodiment, the fixative is UMFIX (see Nadji et al., Appl. Immunohistochem. Mol. Morphol. 13:277-282, 2005). In one embodiment, the fixative is Z7 (see Lykidis et al., Nucleic Acids Res. 35:e85, 2007). In one embodiment, the fixative is ZBF (see Paavilainen et al., Histochem. Cytochem.:Official J. Histochem. Soc. 58:237-246, 2010).

[0202] The kits provided herein include an aldehyde-containing fixative. In one embodiment, the aldehyde-containing fixative in the kit is formaldehyde. In one embodiment, the aldehyde-containing fixative in the kit is glutaraldehyde. In one embodiment, the aldehyde-containing fixative in the kit is Bouin fixative, which is a solution of picric acid, formaldehyde, and acetic acid. In one embodiment, the aldehyde-containing fixative in the kit is a mixture of formaldehyde and glutaraldehyde. In one embodiment, the aldehyde-containing fixative in the kit is FAA, which is a solution of ethanol, acetic acid, and formaldehyde. In one embodiment, the aldehyde-containing fixative in the kit is periodate-lysine-paraformaldehyde (PLP), which is a solution of paraformaldehyde, L-lysine, and INaO4. In one embodiment, the aldehyde-containing fixative in the kit is phosphate-buffered formalin (PBF). In one embodiment, the aldehyde-containing fixative in the kit is formal calcium, which is a solution of formaldehyde and calcium chloride. In one embodiment, the aldehyde-containing fixative in the kit is formal saline solution, which is a solution of formaldehyde and sodium chloride. In one embodiment, the aldehyde-containing fixative in the kit is zinc formalin, which is a solution of formaldehyde and zinc sulfate. In one embodiment, the aldehyde-containing fixative in the kit is Helly fixative, which is a solution of formaldehyde, potassium dichromate, sodium sulfate, and mercury chloride. In one embodiment, the aldehyde-containing fixative in the kit is Hollande fixative, which is a solution of formaldehyde, copper acetate, picric acid, and acetic acid. In one embodiment, the aldehyde-containing fixative in the kit is Gendre solution, which is a solution of formaldehyde, ethanol, picric acid, and glacial acetic acid. In one embodiment, the aldehyde-containing fixative in the kit is alcohol-formalin, which is a solution of formaldehyde, ethanol, and calcium acetate. In another embodiment, the aldehyde-containing fixative in the kit is formalic acid alcohol, which is a solution of formaldehyde, glacial acetic acid, and ethanol.In one embodiment, the aldehyde-containing fixative in the kit is a mixture of fixatives, and at least one of the fixatives in the mixture is formaldehyde or glutaraldehyde. In another embodiment, the aldehyde-containing fixative in the kit is a mixture of fixatives that are not used simultaneously but are used sequentially, and at least one of the fixatives is formaldehyde or glutaraldehyde.

[0203] In some embodiments, the aldehyde-containing fixative in the kit provided herein contains about 5% to about 50% formaldehyde. In other embodiments, the aldehyde-containing fixative contains about 10% to about 40% formaldehyde. In yet another embodiment, the aldehyde-containing fixative contains about 12% to about 37% formaldehyde.

[0204] In some embodiments, the aldehyde-containing fixative in the kit provided herein contains formaldehyde at various concentrations. In one embodiment, the aldehyde-containing fixative contains about 5% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 6% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 7% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 8% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 9% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 10% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 11% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 12% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 13% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 14% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 15% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 16% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 17% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 18% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 19% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 20% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 30% formaldehyde. In one embodiment, the aldehyde-containing fixative contains about 35% formaldehyde. In another embodiment, the aldehyde-containing fixative contains about 40% formaldehyde. In yet another embodiment, the aldehyde-containing fixative contains about 50% formaldehyde.

[0205] 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.

[0206] In some embodiments, the target nucleic acid is DNA. In some embodiments, the target nucleic acid is RNA. In some embodiments, the target nucleic acid is long RNA. In some embodiments, the target nucleic acid is short RNA. In some embodiments, the target nucleic acid is RNA containing fewer than 100 nucleotides. In other embodiments, the target nucleic acid is RNA containing fewer than 50 nucleotides. In other embodiments, the target nucleic acid is RNA containing 15 to 40 nucleotides. In some embodiments, the target nucleic acid is sncRNA. In other embodiments, the target nucleic acid is miRNA, siRNA, piRNA, or ASO. In yet another embodiment, the target nucleic acid is endogenous RNA or exogenous RNA.

[0207] In certain embodiments, the kits provided herein include agents for performing RNAscope®, as described in detail, for example, 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.

[0208] 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.

[0209] In some embodiments, the kit further comprises a signal-generating complex as described in Section 5.3 above, which may include a labeled probe, an amplifier, a pre-amplifier, and / or a pre-pre-amplifier.

[0210] In some embodiments, the kit further includes fixatives and agents for processing the sample in preparation for hybridization, as well as other agents or materials for performing RNA ISH, such as agents for washing the sample.

[0211] 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.).

[0212] The kits provided herein may include labels or inserts. The labels or inserts may include information about the condition, disorder, disease, or symptom for which the kit components may be used. The labels or inserts may include instructions for a clinician or subject to use one or more of the kit components in a method, treatment protocol, or treatment regimen. In some embodiments, the kit can be used for tissue and cell type identification. In some embodiments, the kit can be used for identification of different stages of development. In some embodiments, the kit can be used for the detection of clinical biomarkers of cancer. In some embodiments, the kit can be used to diagnose a disease or disorder based on the expression of one or more denatured small RNAs or the presence of pathogen-derived small RNAs. In some embodiments, the kit can be used for characterizing adult tissues. In some embodiments, the kit can be used for the detection of clinical biomarkers for pathogen diagnosis. In some embodiments, the kit can be used for the detection and characterization of small RNA-based therapies. In some embodiments, the kit can be used for initial efficacy confirmation of small RNA-based therapies. In some embodiments, the kit can be used to continuously monitor the effectiveness of small RNA-based therapies. In some embodiments, the kit can be used to determine the effectiveness of small RNA-based therapies. In some embodiments, the kit can be used to detect, localize, and quantify the presence of siRNA. In some embodiments, the kit can be used to detect, localize, and quantify the presence of ASO molecules. In some embodiments, the kit can be used to detect and identify small RNAs derived from pathogens.

[0213] [Examples] The following describes various methods and materials used in the research and is presented to provide a complete disclosure and explanation of how the present invention is prepared and used, and is not intended to limit the scope of what the inventors consider to be their invention, 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 are not necessarily performed, but rather should be understood as indicating that these descriptions can be performed to produce data, etc., 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.

[0214] 6.1 Integrating post-fixation strategies for small nucleic acid preservation into standard RNA ISH workflows Conventional biological specimens are preserved according to a standard deparaffinization process, which involves heating the sample to dissolve the paraffin wax, followed by a series of xylene and ethanol washes to remove all traces of wax. This process can lead to the loss of low molecular weight nucleic acids. To minimize diffusion-induced loss during sample pretreatment and hybridization / washing, the sample is post-fixed at ambient temperature for a minimum of two hours using an aldehyde-containing fixative before sample pretreatment. Samples post-fixed for less than two hours showed minimal retention of these nucleic acid species. Optimal retention was observed during post-fixation of 2 to 18 hours, depending on the fixative used. Biological specimens post-fixed with formaldehyde concentrations equal to or higher than approximately 12% required only a short (2-hour) exposure to the fixative, while those with formaldehyde concentrations below approximately 12%, such as 10% neutral buffered formalin (NBF), required a longer (18-hour) treatment. Samples following the post-fixation workflow are fully compatible with subsequent steps in the general ISH workflow outlined, including sample preparation, target probe hybridization, and detection.

[0215] 6.2 Post-fixation with formaldehyde at a concentration equal to or higher than 12% for miRNA detection. Post-fixation with formaldehyde minimizes miRNA loss and improves detection sensitivity by RNAscope® ISH. Mouse formalin-fixed paraffin-embedded tissue sections were post-fixed at ambient temperature for 2 hours with untreated or formaldehyde concentrations ranging from approximately 12% to 37% prepared in 1X PBS. Maximum miRNA retention was observed when fixed after 2 hours with a formaldehyde concentration equal to or higher than approximately 12%. Specific miRNAs were detected using RNAscope® 2.5 High Definition (HD)-Red Assay according to established conditions for formalin-fixed paraffin-embedded tissue processing. ISH signals appeared as spots, and hematoxylin stained individual cell nuclei. As shown in Figure 2A, miR-1a-3p, which is abundantly expressed in the heart, was detected after post-fixation. As shown in Figure 2B, miR-132-3p, which is abundantly expressed in the brain, was detected after post-fixation. In the absence of post-fixation, miRNA diffuses from the tissue and is undetectable, as evidenced by the absence of spots in cardiac and brain tissue. In contrast, spot formation was observed in post-fixed tissue, demonstrating the effectiveness of this disclosure. No further advantage in miRNA detection sensitivity was observed, as a similar number of spots were observed throughout the tissue even when the formaldehyde concentration was increased from 12% to 37%.

[0216] 6.3 Post-fixation with 10% NBF for miRNA detection Mouse formalin-fixed paraffin-embedded tissue sections were either left untreated or post-fixed for 18 hours at ambient temperature with 10% NBF, the most commonly used aldehyde-containing fixative with approximately 4% formaldehyde content, prepared in distilled water and sodium phosphate for neutral pH. Shorter incubation times resulted in suboptimal miRNA retention in the tissue. Using RNAscope® 2.5 High Definition (HD)-Red Assay, miR-1a-3p (Figure 3A), miR-132-3p (Figure 3B), miR-122-5p (Figure 3C), and let-7 (Figure 3D), which are abundantly expressed in the heart and brain respectively, were detected according to established conditions for processing formalin-fixed paraffin-embedded tissue. Incorporating a post-fixation step into the ISH assay provides superior detection sensitivity for low molecular weight nucleic acids (indicated as spot formation) compared to untreated tissue.

[0217] 6.4 Post-fixation with formaldehyde for detection of long-chain RNA species The post-fixation workflow has been shown to be fully suitable for detecting long RNA species. Human and mouse formalin-fixed paraffin-embedded tissues were processed either untreated or post-fixed with 10% NBF, and then processed using the general-purpose ISH workflow outlined in Figure 1. As shown in Figure 4A, human TATA box-binding protein (TBP) mRNA was detected in untreated or NBF-post-fixed human cutaneous A431 cells using RNAscope® 2.5 High Definition (HD)-Red Assay. Mouse polymerase RNA II polypeptide A (POLR2A) mRNA was detected in mouse intestinal tissue (Figure 4B) and brain tissue (Figure 4C), respectively, using RNAscope® 2.5 High Definition (HD)-Red Assay. Similar detection sensitivity for long RNA species was observed in both untreated and post-fixed tissues. This suggests that small RNA species may benefit from this alternative ISH workflow, but the detection of long RNA species is not impaired by it.

[0218] 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]

[0219] [Figure 1] This shows the incorporation of post-fixation steps in a standard RNA ISH workflow. Post-fixation steps that are not present in a standard RNA ISH workflow are labeled with a double-bordered box. [Figure 2A]This study demonstrates that post-fixation with formaldehyde concentrations equal to or higher than 12% minimizes miRNA loss and improves detection sensitivity by RNAscope® ISH. Mouse formalin-fixed paraffin-embedded tissue sections were either untreated or post-fixed with formaldehyde concentrations ranging from 12% to 37%. Two miRNAs were detected using RNAscope® 2.5 High Definition (HD)-Red Assay, following established conditions for formalin-fixed paraffin-embedded tissue processing. ISH signals appeared as spots, and hematoxylin stained individual cell nuclei. The figure shows the detection of miR-1a-3p, which is abundantly expressed in the heart, after the post-fixation process. [Figure 2B] This study demonstrates that post-fixation with formaldehyde concentrations equal to or higher than 12% minimizes miRNA loss and improves detection sensitivity by RNAscope® ISH. Formalin-fixed, paraffin-embedded mouse tissue sections were either untreated or post-fixed with formaldehyde concentrations ranging from 12% to 37%. Two miRNAs were detected using RNAscope® 2.5 High Definition (HD)-Red Assay, following established conditions for formalin-fixed, paraffin-embedded tissue processing. ISH signals appear as spots, and hematoxylin stains individual cell nuclei. The figure shows the detection of miR-132-3p, which is abundantly expressed in the brain. [Figure 3A]This study demonstrates that post-fixation using 10% neutral buffered formalin (NBF) with approximately 4% formaldehyde content minimizes miRNA loss and improves detection sensitivity by RNAscope® ISH. Mouse formalin-fixed paraffin-embedded tissue sections were either untreated or post-fixed with 10% NBF. Multiple miRNAs were detected using RNAscope® 2.5 High Definition (HD)-Red Assay, following established conditions for formalin-fixed paraffin-embedded tissue processing. ISH signals appeared as spots, and hematoxylin stained individual cell nuclei. The figure shows the detection of miR-1a-3p, which is abundantly expressed in the heart, after the post-fixation process. [Figure 3B] This study demonstrates that post-fixation using 10% neutral buffered formalin (NBF) with approximately 4% formaldehyde content minimizes miRNA loss and improves detection sensitivity by RNAscope® ISH. Mouse formalin-fixed paraffin-embedded tissue sections were either untreated or post-fixed with 10% NBF. Multiple miRNAs were detected using RNAscope® 2.5 High Definition (HD)-Red Assay, following established conditions for formalin-fixed paraffin-embedded tissue processing. ISH signals appeared as spots, and hematoxylin stained individual cell nuclei. The figure shows the detection of miR-132-3p, which is abundantly expressed in the brain, after the post-fixation process. [Figure 3C]This study demonstrates that post-fixation using 10% neutral buffered formalin (NBF) with approximately 4% formaldehyde content minimizes miRNA loss and improves detection sensitivity by RNAscope® ISH. Mouse formalin-fixed paraffin-embedded tissue sections were either untreated or post-fixed with 10% NBF. Multiple miRNAs were detected using RNAscope® 2.5 High Definition (HD)-Red Assay, following established conditions for formalin-fixed paraffin-embedded tissue processing. ISH signals appeared as spots, and hematoxylin stained individual cell nuclei. The figure shows the detection of miR-122-5p, which is abundantly expressed in liver, after the post-fixation process. [Figure 3D] This study demonstrates that post-fixation with 10% neutral buffered formalin (NBF) containing approximately 4% formaldehyde minimizes miRNA loss and improves detection sensitivity by RNAscope® ISH. Mouse formalin-fixed paraffin-embedded tissue sections were either left untreated or post-fixed with 10% NBF. Multiple miRNAs were detected using RNAscope® 2.5 High Definition (HD)-Red Assay, following established conditions for formalin-fixed paraffin-embedded tissue processing. ISH signals appeared as spots, and hematoxylin stained individual cell nuclei. The figure shows the detection of eccentrically expressed let-7 after the post-fixation process. [Figure 4A] The post-fixation workflow demonstrated complete suitability for detecting long RNA species. Human and mouse formalin-fixed paraffin-embedded tissues were either left untreated or post-fixed with 10% NBF and processed using the general-purpose ISH workflow outlined in Figure 1. The figure shows the detection of human TATA box-binding protein (TBP) mRNA in human cutaneous A431 cells using RNAscope® 2.5 High Definition (HD)-Red Assay. [Figure 4B]The post-fixation workflow demonstrated complete suitability for the detection of long-chain RNA species. Human and mouse formalin-fixed paraffin-embedded tissues were either left untreated or post-fixed with 10% NBF and processed using the general-purpose ISH workflow outlined in Figure 1. The figure shows the detection of mouse polymerase RNA II polypeptide A (POLR2A) mRNA in mouse intestinal samples. [Figure 4C] The post-fixation workflow demonstrated complete suitability for detecting long-chain RNA species. Human and mouse formalin-fixed paraffin-embedded tissues were either left untreated or post-fixed with 10% NBF and processed using the general-purpose ISH workflow outlined in Figure 1. The figure shows the detection of mouse polymerase RNA II polypeptide A (POLR2A) mRNA in brain tissue.

Claims

1. A method for preparing a biological sample for in situ hybridization, i. A first fixation step, which includes fixing the biological sample with a drug, ii. A post-fixation step comprising fixing the biological sample with a formaldehyde-containing fixative at ambient temperature for 2 to 18 hours after the first fixation step and before the in situ hybridization, wherein the formaldehyde-containing fixative contains 12% to 37% formaldehyde. The in situ hybridization method is a method for detecting nucleic acids containing 100 or fewer nucleotides in the biological sample.

2. The method according to claim 1, wherein the in situ hybridization is RNA in situ hybridization or DNA in situ hybridization.

3. The method according to claim 2, wherein the RNA in situ hybridization detects RNA containing fewer than 50 nucleotides.

4. The method according to claim 2, wherein the RNA in situ hybridization detects RNA containing 15 to 40 nucleotides.

5. The method according to claim 2, wherein the RNA in situ hybridization detects small non-coding RNA (sncRNA).

6. The method according to claim 2, wherein the RNA in situ hybridization or DNA in situ hybridization detects microRNA (miRNA), small interfering RNA (siRNA), PIWI-interacting RNA (piRNA), or antisense oligonucleotide (ASO) molecules, and the RNA in situ hybridization or DNA in situ hybridization detects endogenous or exogenous nucleic acids.

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 comprises cultured cells or exosomes.

11. The method according to any one of claims 1 to 10, wherein the agent in the first fixation step is selected from the group consisting of formaldehyde, glutaraldehyde, methanol, ethanol, and acetone.

12. The method according to any one of claims 1 to 10, wherein the post-fixation step includes fixing the biological sample with the formaldehyde-containing fixative for two hours.

13. The method according to any one of claims 1 to 12, wherein the formaldehyde-containing fixing solution in the post-fixation step comprises an additional agent in addition to formaldehyde.

14. A method for detecting target nucleic acids in cells, (i) preparing a biological sample by the method described in any one of claims 1 to 13, and (ii) providing at least one set of one or more target probes capable of hybridizing to the target nucleic acid. (iii) 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. (iv) Hybridizing the target nucleic acid with the set of one or more target probes, (v) capturing the signal-generating complex on the set of one or more target probes, thereby capturing the signal-generating complex on the target nucleic acid, The method wherein the target nucleic acid comprises less than 100 nucleotides.

15. The method according to claim 14, wherein each of the 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-generating complex, the T section of one or more target probes being complementary to a non-overlapping region of the target nucleic acid, and the L section of one or more target probes being complementary to a non-overlapping region of the nucleic acid component of the signal-generating complex.

16. The method according to claim 14 or 15, wherein the target nucleic acid is RNA containing fewer than 50 nucleotides.

17. The method according to claim 14 or 15, wherein the target nucleic acid is RNA containing 15 to 40 nucleotides.

18. The method according to claim 14 or 15, wherein the target nucleic acid is a small non-coding RNA (sncRNA).

19. The method according to claim 14 or 15, wherein the target nucleic acid is a microRNA (miRNA), a small interfering RNA (siRNA), a PIWI-interacting RNA (piRNA), or an antisense oligonucleotide (ASO) molecule, and the RNA in situ hybridization or DNA in situ hybridization is for detecting endogenous or exogenous nucleic acids.

20. The method according to any one of claims 1 to 19, used for identifying a tissue or cell type.

21. A method according to any one of claims 1 to 19, used to determine the stage of occurrence.

22. A method according to any one of claims 1 to 19, used for characterizing adult tissue.

23. A method for preserving a target nucleic acid in a pre-fixed sample for in situ detection of the target nucleic acid, comprising a post-fixation step of applying a formaldehyde-containing fixative to the sample for 2 to 18 hours before performing an in situ hybridization detection assay using a set of one or more probes that hybridize to the target nucleic acid, wherein the formaldehyde-containing fixative contains 12% to 37% formaldehyde, and the target nucleic acid contains fewer than 100 nucleotides.

24. A method for in-situ detection of target nucleic acids in a pre-fixed sample, (i) A post-fixation step comprising applying a formaldehyde-containing fixative to the pre-fixed sample at ambient temperature for 2 to 18 hours, wherein the formaldehyde-containing fixative contains 12% to 37% formaldehyde, (ii) The step of performing an in situ hybridization detection assay using a set of one or more probes that hybridize to the target nucleic acid, The method wherein the target nucleic acid comprises less than 100 nucleotides.

25. The method according to claim 23 or claim 24, wherein the sample is a tissue sample or derived from a tissue sample.

26. The method according to claim 23 or claim 24, wherein the sample is a blood sample or derived from a blood sample.

27. The method according to claim 23 or claim 24, wherein the sample is a cytological sample or is derived from a cytological sample.

28. The method according to claim 23 or claim 24, wherein the sample comprises cultured cells or exosomes.

29. The method according to any one of claims 23 to 28, wherein the post-fixation step includes fixing the sample with the formaldehyde-containing fixative for two hours.

30. The method according to any one of claims 23 to 29, wherein the formaldehyde-containing fixing solution in the post-fixation step comprises an additional agent in addition to formaldehyde.

31. The set of probes includes one or more target probes that can hybridize to the target nucleic acid, and the method is (a) 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. (b) Hybridizing the target nucleic acid with the set of one or more target probes, (c) The method according to any one of claims 23 to 30, further comprising capturing the signal-generating complex on the set of one or more target probes, thereby capturing the signal-generating complex on the target nucleic acid.

32. The method according to claim 31, wherein each of the 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-generating complex, the T section of one or more target probes being complementary to a non-overlapping region of the target nucleic acid, and the L section of one or more target probes being complementary to a non-overlapping region of the nucleic acid component of the signal-generating complex.

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