Method for antigen retrieval of immobilized samples for multiple applications

Abstract

The techniques provided herein relate to a method for antigen retrieval of fixed samples, particularly freeze-fixed tissue samples, by using a two-step demasking approach, in which the biological sample is treated at low temperatures (50-75°C) with different buffers.

Description

Technical Field

[0001] The technology provided in this specification relates to a method for antigen activation of fixed samples, particularly cryo-fixed tissue samples, by using a two-step de-masking approach, in which a biological sample is treated at a low temperature (50 - 75 °C) with different buffers.

Background Art

[0002] In the fields of histology, pathology, and cell biology, fixation is the preservation of biological tissues from degradation by autolysis or putrefaction. It may also terminate any ongoing biochemical reactions and enhance the mechanical strength or stability of the treated tissue. Tissue fixation is an important step in the preparation of histological sections, and its broad objective is to perform this by a method that preserves cells and tissue components and enables the preparation of thin stained sections. This makes it possible to examine the structure of the tissue, which is determined by the shape and size of macromolecules such as proteins and nucleic acids (present inside and around cells).

[0003] Due to the nature of tissue processing, samples are required to be "fixed" prior to embedding in paraffin and microtoming with a microtome to produce tissue sections suitable for immunostaining. In this process, proteins are preserved using formaldehyde treatment, which produces chemical cross-links that retain the cellular characteristics of the tissue. Formaldehyde mainly preserves or fixes tissues or cells by cross-linking primary amine groups in proteins to other neighboring nitrogen atoms in proteins or DNA via -CH2- linkages. However, the process of tissue fixation often masks antigens on specific proteins that are desirable to detect for diagnostic and prognostic purposes. Typically, these procedures are optimized for the detection of individual target molecules, and if necessary, serial sections are treated in different ways for the detection of additional target molecules.

[0004] With advancements in the ability to detect multiple antigens within a single sample, uniform tissue processing that is compatible with the detection of multiple proteins is required. Non-patent document 1 discloses heating a formaldehyde-fixed sample in a series of two Tris-HCl buffers. Patent document 1 discloses antigen retrieval by heating in a low pH buffer, while non-patent document 2 discloses microwave heating of a formaldehyde-fixed sample in different antigen retrieval solutions.

[0005] A technique based on Gerdes et al. (Patent Document 2) is a method for antigen retrieval of a formaldehyde-fixed tissue sample, the step of which includes incubating the formaldehyde-fixed tissue sample in a first antigen retrieval solution, transferring the tissue sample to a second antigen retrieval solution, and incubating the tissue sample in the second antigen retrieval solution.

[0006] This system is designed to demask a target epitope on formalin-fixed paraffin-embedded tissue at temperatures exceeding 90°C. The formalin-fixed paraffin-embedded tissue is incubated in a first antigen retrieval solution (pH 5-7). The tissue sample is then transferred to a second antigen retrieval buffer (pH 7.5-11) without further processing. The first and second antigen retrieval buffers may be interchanged. Incubation is performed for more than 10 minutes, preferably for a period of about 20 minutes. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent Application Publication No. 2004 / 035364 [Patent Document 2] U.S. Patent No. 8067241 [Non-patent literature]

[0008] [Non-Patent Document 1] S Yamashita et al, J Histochem Cytochem, 2005, Vol. 53, p. 13-21 [Non-Patent Document 2] SR Shi et al,J Histochem Cytochem,Vol.39,p.741-748 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] Several known methods for detecting analytes in the art have many drawbacks. Against this backdrop, the underlying objective of this disclosure is to provide a method that can reduce or even avoid the drawbacks of prior art methods. [Means for solving the problem]

[0010] This disclosure relates to a novel cryogenic sample preparation procedure for in situ detection of biomolecules in fixed biological samples, such as formaldehyde-fixed biological samples.

[0011] Cross-linking can alter the biochemical properties of biomolecules, rendering them unable to bind to specific binding molecules. This is caused by intramolecular and intermolecular cross-linking of biomolecules by formaldehyde, alteration of biomolecular conformation, steric hindrance between biomolecules and specific binding molecules, and masking of the binding sites of specific binding molecules. A two-step demasking approach, in which biological samples are treated at low temperatures (50-75°C) and sequentially with weakly acidic buffer (pH 6.5-7) and weakly basic buffer (pH 8-10) for a set period (0.5-2 hours each), reduces masking of biomolecules. This restores the ability to form specific intermolecular bonds. Heating may be performed using a water bath, hot plate, microwave, or thermal cycler to provide uniform heating to tissue samples immersed in the demasking solution. The presence of biomolecules in biological samples can be identified by histological techniques such as in situ hybridization and immunohistochemistry, thereby facilitating multi-omics analysis. These two histological techniques can be performed sequentially on the same slide by applying the proposed demasking method.

[0012] However, this approach is not limited to biological samples fixed with crosslinking agents, but is also applicable to biological samples fixed in coagulation agents (e.g., alcohol and acetone), biological samples fixed in formaldehyde and embedded in paraffin (FFPE), and cryopreserved biological samples.

[0013] In a first aspect, the present disclosure relates to a method for antigen retrieval of a fixed sample, the method comprising the following steps: a) Incubate the fixed sample in the first antigen retrieval solution at a temperature of 50-75°C, b) Transferring the fixed sample to the second antigen retrieval solution, c) Incubate the fixed sample in the second antigen retrieval solution at a temperature of approximately 50-75°C. Includes, Here, the first antigen retrieval solution contains a buffer having a pH range of approximately 6.5 to 7, and the second antigen retrieval solution contains a buffer having a pH range of approximately 8 to 10, or The first antigen retrieval solution contains a buffer with a pH range of approximately 8 to 10, and the second antigen retrieval solution contains a buffer with a pH range of approximately 6.5 to 7.

[0014] Before describing this disclosure in detail, please understand that this disclosure is not limited to any specific component part of the steps of the methods described. Also, please understand that the terms used herein are intended to describe only specific embodiments and are not intended to limit them. Furthermore, please note that the singular forms “a,” “an,” and “the” used herein and in the appended claims include singular and / or plural meanings unless the context clearly indicates otherwise. Additionally, where a numerically delimited parameter range is given, please understand that the range includes these limits. [Brief explanation of the drawing]

[0015] [Figure 1] This is an overview of an embodiment of a two-step demasking process on a fixed tissue for multi-omics analysis. [Figure 2] Frozen sections of mouse kidney tissue probed for lysozyme, MDM2, and p21 are shown. Rabbit primary antibodies against lysozyme (BioLegend, #860001), and mouse primary antibodies against MDM2 (Santa Cruz, sc-965) and p21 (Origene, AM09100PU-T) were incubated on tissue pre-treated by a two-step demasking method. After incubation of the primary antibodies, Alexa Fluor 647 conjugated to goat anti-rabbit IgG (Abcam, ab150083) was added. Images were acquired using a ZEISS Celldiscoverer 7 with an Apochromat 5X / 0.35 objective lens and a 2X tube lens. [Figure 3]Further shown are frozen sections of tissues from a colorectal xenograft model by using the method for antigen activation according to the present disclosure.

Mode for Carrying Out the Invention

[0016] The present disclosure features a method for antigen activation in fixed samples, which is carried out sequentially. The method is suitable for fixed tissues (cryo-fixed or formalin-fixed), those stored at -80°C or those stored by embedding the tissue in paraffin.

[0017] Cryo-fixed tissues can be post-fixed with a coagulant fixative (alcohol or acetone) or a cross-linking fixative (formaldehyde). As described above, the present disclosure relates to a two-step demasking approach, in which biological samples are treated continuously for a certain period (0.5 - 2 hours for each step) at a low temperature (50 - 75°C) with a weakly acidic buffer (pH 6.5 - 7) and a weakly basic buffer (pH 8 - 10) to reduce the masking of biomolecules. Thereby, the ability to form specific bonds between molecules is restored. Heating can be performed using a water bath, hot plate, microwave, or thermal cycler to provide uniform heating to the tissue samples immersed in the demasking solution.

[0018] The presence of biomolecules in biological samples can be identified by histological techniques such as in situ hybridization and immunohistochemistry, which promotes multi-omics analysis. These two histological techniques can be carried out continuously on the same slide by applying the proposed demasking method.

[0019] This approach is not limited to biological samples fixed with a cross-linking fixative, but is also applicable to biological samples fixed with a coagulant fixative (such as alcohol and acetone), biological samples fixed with formaldehyde and embedded in paraffin (FFPE), and cryo-fixed biological samples.

[0020] Surprisingly, the antigen retrieval process in the prior art (U.S. Patent No. 8,067,241) was found to be too hot and unsuitable for cryopreserved tissues. High-temperature incubation can damage tissue structure and morphology, or cause tissue to detach from the holder. Furthermore, this technique is only applicable to tissues fixed with formaldehyde and embedded in paraffin. High-temperature processing (above 80°C) is also unsuitable for instruments used in spatial biology analysis platforms, and is particularly unsuitable when in situ hybridization and immunofluorescence are performed on the same tissue sample.

[0021] The analysis and detection of trace analytes such as RNA or proteins in biological and non-biological samples are routinely performed in clinical and analytical settings. Numerous analytical methods have been established for this purpose. Some of these assign a distinct, readable code to a specific first analyte, using a different encoding technique than the code assigned to a specific second analyte.

[0022] One of the prior art techniques in this field is so-called "single-cell fluorescence in situ hybridization" (smFISH), which was essentially developed to detect mRNA molecules in a sample. In Lubeck et al. (2014), Single-cell in situ RNA profiling by sequential hybridization, Nat. Methods 11(4), pp. 360-361, the target mRNA is detected via a specific, directly labeled probe set. After one round of hybridization and detection, the mRNA-specific probe set is eluted from the mRNA, and the same probe set with other (or identical) fluorescent labels is used in subsequent rounds of hybridization and imaging, generating a gene-specific color scheme across multiple rounds. This technique requires probe sets with multiple different tags for each transcript, and these probe sets must be denatured after each detection round.

[0023] Further developments of this technology do not use directly labeled probe sets. Instead, the oligonucleotides in the probe set provide nucleic acid sequences that function as initiators for hybridization chain reactions (HCRs), which is a technique that enables signal amplification. See Shah et al. (2016), In situ transcription profiling of single cells reveals spatial organization of cells in the mouse hippocampus, Neuron 92(2), pp. 342-357.

[0024] Immunohistochemistry (IHC) refers to the process of detecting, localizing, and / or quantifying antigens, such as proteins, in biological samples using antibodies specific to a particular antigen. IHC offers the significant advantage of being able to precisely identify where a particular protein is located within a tissue sample. This is also an effective means of analyzing the tissue itself. In situ hybridization (ISH) refers to the process of detecting, localizing, and quantifying nucleic acids. Both IHC and ISH can be performed on a variety of biological samples, including tissues (e.g., fresh-frozen, formalin-fixed, paraffin-embedded) and cytological samples. Target recognition can be detected using various labels (e.g., chromogenic, fluorescent, luminescent, radioactive), regardless of whether the target is a nucleic acid or an antigen. Amplification of recognition events is desirable for reliably detecting, localizing, and quantifying targets in a clinical setting, and the ability to reliably detect low-expression cellular markers is becoming increasingly important for diagnostic purposes. For example, by depositing hundreds or thousands of label molecules on a marker site in response to a single antigen detection event, the ability to detect that recognition event through amplification is improved.

[0025] According to this disclosure, “antigen” is a target whose presence or absence in a sample is specifically detected, and if present, includes the encoding of it. It can be any kind of entity, such as a protein, polypeptide, protein, or nucleic acid molecule (e.g., RNA, PNA, or DNA). The analyte provides at least one site for specific binding to an antibody, antibody fragment, and / or aptamer.

[0026] An "antigen" specifically refers to a substance that can bind to antibodies, antibody fragments, and / or aptamers. Antigens can be endogenous, in which case they are produced within cells as a result of normal or abnormal cellular metabolism, or due to viral or intracellular bacterial infection. Endogenous antigens include heterologous antigens, autologous antigens, and idiotypic or homologous antigens. Antigens may also be tumor-specific antigens or may be presented by tumor cells. In this case, they are called tumor-specific antigens (TSAs) and are generally due to tumor-specific mutations. Antigens may also be tumor-associated antigens (TAAs), which are presented by tumor cells and normal cells. Antigens also include CD antigens, which refer to one of a number of cell surface markers expressed by leukocytes that can be used to distinguish cell lineages or developmental stages. Such markers can be identified by specific monoclonal antibodies and numbered by differentiation clusters.

[0027] In particular, the antigen may be detected or bound by an antibody, antibody fragment, aptamer, or nucleic acid that specifically interacts with and binds to the analyte, and the antibody fragment in particular is selected from the group consisting of Fab, scFv, single domain, bis scFv, Fab2, Fab3, minibody, diabody, triplebody, tetrabody, and tandem antibody (tandab).

[0028] "FISH" and "CISH" refer to fluorescence in situ hybridization and chromogenic in situ hybridization, respectively. FISH is a cytogenetic technique used to detect and localize specific DNA sequences on chromosomes, or RNA sequences at transcription sites, as well as their presence or absence in other parts of the cell. FISH uses fluorescent probes that bind only to chromosomal regions showing high sequence homology. CISH uses conventional enzymatic reactions under a bright-field microscope on formalin-fixed paraffin-embedded (FFPE) tissue to detect gene amplification, chromosomal translocations, and chromosome number.

[0029] "Immunostaining" refers to antibody-based and / or aptamer-based methods for detecting specific proteins in a sample. Immunostaining includes both immunocytochemical and immunohistochemical staining. Immunocytochemical (ICC) staining refers to a technique that uses antibodies that target antigens on cells. This may be performed to determine the presence of certain diseases, such as various types of cancer. Immunohistochemical (IHC) staining refers to staining and localizing antigens in tissue sections using labeled antibodies as specific reagents, via antigen-antibody interactions visualized by markers such as fluorescent dyes, reactive enzyme substrates, radioactive elements, or colloidal gold.

[0030] As used herein, “sample” refers to a composition in liquid or solid form that is presumed to contain the coded analyte. In particular, the sample is a biological sample, preferably comprising biological tissue, and more preferably comprising biological cells and / or extracts and / or parts of cells. For example, the cells are prokaryotic or eukaryotic cells, and in particular mammalian cells, especially human cells. In some embodiments, the biological tissue, biological cells, extracts and / or parts of cells are immobilized. In particular, the analyte is immobilized in a permeabilized sample, such as a sample containing cells.

[0031] As used in this disclosure, “cells,” “cell lines,” and “cell cultures” are interchangeable, and all these terms include progeny cells. Therefore, the terms “transformed” or “transformed cells” include the primary target cells and cultures derived therefrom, regardless of the number of passages. It is also understood that all progeny cells may not have exactly the same DNA content due to intentional or accidental mutations. This includes mutant progeny cells that have the same functionality as those screened in the original transformed cells.

[0032] In some advantageous embodiments, the fixed sample is a biological sample, preferably comprising biological tissue of human, animal, or plant origin, and more preferably comprising biological cells and / or extracts and / or cell portions. In particular, the cells are prokaryotic or eukaryotic cells, animal cells, plant cells, and especially cells derived from vertebrate cells, especially mammalian cells, especially human cells.

[0033] Fixation of biological specimens can be carried out with all fixatives known to those skilled in the art, particularly with acids, alcohols, ketones, or other organic substances, such as glutaraldehyde or formaldehyde, formaldehyde-fixed biological specimens, and aqueous formaldehyde solutions containing 1-35 wt.%, preferably 2-10 wt.%, of formaldehyde. According to one embodiment of the method of this disclosure, a biological specimen fixed with formaldehyde and embedded in paraffin is used as the fixed specimen. Such specimens are generally called FFPE specimens. Such FFPE specimens are preferably prepared by first dehydrating a formalin-fixed biological specimen, preferably by an ascending alcohol series, i.e., a series of water / alcohol mixtures with increasing alcohol concentration (finally adding pure alcohol). C-C alcohols are particularly preferred as alcohols, further preferred are ethanol, methanol, and isopropanol, with ethanol being the most preferred. The dehydrated specimen is then immersed in liquid paraffin, and the paraffin is cured after the specimen has sufficiently penetrated the paraffin. With a suitable cutting device, such as a microtome, tissue sections can be produced from a paraffin block, and these sections typically have a thickness of about 5–20 μm for optical microscopy observation.

[0034] In advantageous embodiments, the fixed sample is a cryopreserved tissue sample. For example, the fixed sample is a cryopreserved tissue section. Cryopreserved sections are prepared quickly and relatively easily before fixation and provide an excellent system for visualizing the fine structure of cells. Although cryopreserved sections are less physically stable than paraffin-embedded or resin-embedded sections, they are generally superior in preserving antigenicity and therefore in detecting antigens by microscopy. Preparation of cryopreserved sections does not involve the dehydration step typical of other sectioning methods, and furthermore, sectioning, labeling, and observation of the specimens can usually be carried out in one day. Generally, the sample is rapidly frozen in isopentane or liquid nitrogen. (Small samples such as cells and small tissues may be mixed in a slurry of an inert support medium such as an optimal cutting temperature (OCT) compound before freezing). Rapid freezing reduces ice crystal formation and minimizes morphological damage. Cryopreserved sections can be used in a variety of techniques, including immunochemistry, enzyme detection, and in situ hybridization. In some advantageous embodiments, the fixed sample is a frozen formaldehyde-fixed biological sample, and in particular a formaldehyde-fixed frozen section of tissue.

[0035] In some advantageous embodiments, the sample is stored at -80°C before antigen retrieval / demasking of the biomolecule.

[0036] In some embodiments, the fixed sample is a formaldehyde-fixed biological sample, and more particularly, the formaldehyde-fixed biological sample is a formaldehyde-fixed and paraffin-embedded biological sample. In some embodiments, at least a portion of the paraffin is removed before the sample is brought into contact with a first antigen retrieval solution (first buffer). In favorable examples, the sample is fixed with formaldehyde and embedded in paraffin (FFPE).

[0037] In favorable cases, the sample is a cryopreserved tissue sample, such as a frozen section of tissue, which is post-fixed with a coagulating fixative such as alcohol or acetone, and / or a cross-linking fixative such as formaldehyde.

[0038] In the “incubation” process as understood herein, each part or object, such as a sample or probe, is brought into contact with each other under conditions described herein or conditions well known to those skilled in the art (e.g., pH, temperature, salt conditions, etc.) that enable a chemical reaction, such as demasking an antigen in the immobilized sample. Therefore, such a process may preferably be carried out in a liquid environment, such as a buffer system.

[0039] As stated above, this disclosure also relates to a method for demasking biomolecules in an immobilized sample, the method comprising the following steps: a) Incubate the immobilized sample in the first buffer at a temperature of 50-75°C, b) Transferring the immobilized sample to the second buffer solution, c) Incubate the immobilized sample in the second buffer at a temperature of approximately 50-75°C, Includes, Here, the first buffer solution has a pH range of approximately 5-7, and the second buffer solution has a pH range of approximately 8-10, or The first buffer solution has a pH range of approximately 8-10, and the second antigen buffer solution has a pH range of approximately 5-7.

[0040] In one embodiment of this disclosure, biological tissues and / or biological cells are fixed. Techniques for fixing cells are known to those skilled in the art. In non-limiting examples, cells may be fixed using chemicals such as formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, methanol, acetone, or acetic acid.

[0041] The present invention generally includes embodiments of methods applicable to analytical, diagnostic, or prognostic applications, including detection of analytes, histochemistry, immunostaining, immunohistochemistry, immunocytochemistry, or immunofluorescence. In some embodiments, the methods disclosed herein may be particularly applicable to immunohistochemistry and immunocytochemistry.

[0042] A method is described, according to one embodiment, in which tissue sections derived from pathological sampling are processed prior to protein detection for biomarker evaluation. In one embodiment, the method includes a two-step procedure that is applicable to multiple protein antigens and can provide advanced antigen retrieval. In certain embodiments, this enables multiplexing of clinically relevant samples.

[0043] In some embodiments, the tissue sample includes tissue sections derived from healthy or diseased tissue (e.g., tissue sections from the colon, mammary gland, or prostate). The tissue sample may include a portion or a piece of a tissue section, for example, a thin slice of tissue or cells cut from a tissue section. In some embodiments, sections of the same tissue sample may be analyzed at both the morphological and molecular levels.

[0044] In some embodiments, tissue samples may be sectioned by first fixing them, then dehydrating them with an ascending series of alcohols, and finally immersing and embedding them in paraffin or other sectioning media. In an alternative embodiment, the tissue sample may be sectioned and then fixed. In some embodiments, the tissue sample may be embedded and processed in paraffin. Examples of paraffin that may be used include, but are not limited to, Paraplast, Broloid, and Tissuecan. After the tissue sample is embedded, it may be sectioned into sections by microtome. The thickness of the sections may vary depending on the type of tissue and the analysis. In certain embodiments, the sections may have a preferred thickness in the range of about 2 μm to about 5 μm.

[0045] After sectioning, the sections can be attached to a slide using an adhesive. Examples of slide adhesives include, but are not limited to, silane, gelatin, and poly-L-lysine. In embodiments where paraffin is used as the embedding material, the tissue sections can be deparaffinized and rehydrated in water. The tissue sections can be deparaffinized using, for example, an organic agent (such as xylenes or a series of alcohols).

[0046] In other embodiments, formaldehyde-immobilized tissue samples may be bonded to a solid support to enable analysis, transfer, and movement during the preparation and imaging processes. Tissue samples may be immobilized on the solid support by physicoadsorption, covalent bond formation, or a combination thereof. The solid support may include polymer materials, glass materials, or metallic materials. Examples of solid supports include membranes, microtiter plates, beads, filters, test strips, slides, coverslips, and test tubes.

[0047] In one embodiment, a method is described in which a fixed sample, such as a formaldehyde-fixed tissue sample, is brought into contact with a first antigen retrieval solution and heated to a temperature of 50 to 75°C for at least 0.5 hours, particularly for about 0.5 to about 2 hours, more preferably to a temperature of about 70°C, particularly for about 0.5 hours, more preferably to a temperature of about 70°C, particularly for about 0.5 hours.

[0048] Heating may be carried out using a pressure cooker, oven, heating stand, autoclave, water bath, hot plate, microwave, or steam heater to provide uniform heating to the tissue sample immersed in the antigen retrieval solution.

[0049] Subsequently, the tissue sample was transferred to a second antigen retrieval solution without any further processing, particularly after standing. This solution was preheated to a temperature of 50–75°C for at least 0.5 hours, particularly for about 0.5–2 hours, and more preferably to about 70°C, particularly for about 0.5 hours.

[0050] Preheating can be performed using a pressure cooker, autoclave, water bath, hot plate, microwave, steam heating, or a combination thereof, and can also be performed when heating the first antigen retrieval solution. Preferably, incubation of the sample in the second antigen retrieval solution is performed under atmospheric pressure and solely by immersion in the heated solution. This can prevent tissue damage.

[0051] In one embodiment, the first antigen retrieval solution is a buffer having a pH range of about 5 to about 7, particularly about 6 to about 7. The first antigen retrieval solution may be a commonly used buffer used to maintain the pH in a slightly acidic to neutral range. In certain embodiments, the buffer may include citric acid, potassium dihydrogen phosphate, diethyl barbiturate, piperazine-N,N'-bis(2-ethanesulfonic acid), dimethylarsinic acid, 2-(N-morpholino)ethanesulfonic acid, or a combination thereof. In other embodiments, the buffer may be sodium citrate phosphate buffer.

[0052] In one embodiment, the second antigen retrieval solution is a buffer having an alkaline pH in the range of about 8 to about 10. The second antigen retrieval solution may be a commonly used buffer used to maintain the pH in a slightly alkaline range. In certain embodiments, the buffer may include tris(hydroxymethyl)methylamine (TRIS), boric acid, sodium tetraborate, 2-(N-morpholino)ethanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-tris(hydroxymethyl)methylglycine (Tricine), 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), or a combination thereof. In another embodiment, the buffer may be a TRIS-HCl buffer having a pH of approximately 10 under high temperature conditions.

[0053] In particular, the first antigen retrieval solution is generally defined as a buffer for the first demasking step and may have a pH value in the range of 5-7 or 8-10. In particular, the second antigen retrieval solution is generally defined as a buffer for the second demasking step. Buffer B may have a pH value in the range of 5-7 or 8-10.

[0054] In other embodiments, it should be understood that the first antigen retrieval solution may be a buffer in the range of about 8 to about 10, and the second antigen retrieval solution may be a buffer in the range of about 5 to about 7, particularly in the range of about 6 to about 7, and particularly in the range of 6.5 to 7.

[0055] In summary, this disclosure relates to a two-step demasking approach in which a biological sample is successively treated at low temperatures (50–75°C) with weakly acidic buffer (pH 5–7) and weakly basic buffer (pH 8–10) for a set period (0.5–2 hours each step) to reduce the masking of biomolecules. This restores the ability to form specific bonds between molecules. Heating can be performed using a water bath, oven, stove, hot plate, microwave, or thermal cycler to provide uniform heating to the tissue sample immersed in the demasking solution. The presence of biomolecules in the biological sample can be identified by histological techniques such as in situ hybridization and immunohistochemistry, thereby facilitating multi-omics analysis. These two histological techniques can be performed successively on the same slide by applying the proposed demasking method.

[0056] Antigens activated by exposure to first and second antigen retrieval solutions may become more sensitive to immunostaining to enable both analytical and functional morphological studies. Immunostaining includes both immunohistochemical (IHC) and immunocytochemical (ICC) staining. In certain embodiments, improvements may include increased positive staining intensity and reduced background staining.

[0057] Therefore, the first or second antigen retrieval solution is citric acid, trisodium citrate, phosphoric acid, 2-(N-morpholino)ethanesulfonic acid monohydrate, bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane, N-(2-acetamide)iminodiacetic acid, N-(carbamoylmethyl)iminodiacetic acid, N-(2-acetamide)-2-aminoethanesulfonic acid, 1,4-piperazinedietanesulfonic acid, 3-morpholino-2-hydroxypropanesulfonic acid, 1,3- It may contain bis[tris(hydroxymethyl)methylamino]propane, 3-(N-morpholino)propanesulfonic acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, imidazole, 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, or tris(hydroxymethyl)aminomethane. 4-(N-morpholino)butanesulfonic acid, piperazine-1,4-bis(2-hydroxypropanesulfonic acid) dihydrate, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, N-[tris(hydroxymethyl)methyl]glycine, diglycine, N,N-bis(2-hydroxyethyl)glycine, N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid), N-[tris(hydroxymethyl)methyl]-3- Aminopropanesulfonic acid, 2-amino-2-methyl-1,3-propanediol, N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, 2-(cyclohexylamino)ethanesulfonic acid, 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid, boric acid, sodium tetraborate, ethylenediaminetetraacetic acid, sodium bicarbonate, sodium carbonate, and nonionic surfactants.

[0058] In certain embodiments, immunostaining of the sample may be performed after application of a second antigen retrieval solution. The antibody solution (e.g., probe) may be in contact with the tissue section for a sufficient time under conditions suitable for the labeled antibody to bind to the antigen. Two detection methods may be used: direct or indirect. In direct detection, a primary antibody labeled with a signal generator (e.g., a fluorescently labeled primary antibody) may be incubated with the antigen in the tissue sample. This antigen may be visualized without further antibody interaction. In indirect detection, an unbound primary antibody may be incubated with the antigen, after which a labeled secondary antibody may bind to the primary antibody. Signal amplification may occur because multiple secondary antibodies may react with different epitopes on the primary antibody. In embodiments where the secondary antibody can bind to an enzyme label, a chromogenic or fluorescent substrate may be added to visualize the antigen. In some embodiments, two or more (up to four) primary antibodies (labeled or unlabeled) may be in contact with the tissue sample. The unlabeled antibody may be in contact with its corresponding labeled secondary antibody. In some embodiments, other methods for signal enhancement may be employed, such as the use of labeled tertiary or quaternary antibodies.

[0059] In some embodiments, a nucleic acid probe is applied to the sample after an antigen retrieval process to perform fluorescence in situ hybridization (FISH) or chromogenic in situ hybridization (CISH).

[0060] This method is further developed to the extent that the encoded analyte can be detected by any means adapted to visualize the signal element. Examples of detectable physical features include, for example, light, chemical reactions, molecular weight, and radioactivity.

[0061] In some advantageous embodiments, the signal generated by the signal element, i.e., the signal generated by the binding of the signal oligonucleotide to the decoding oligonucleotide that interacts with the corresponding analyte probe bound to each analyte, is determined by the following: (a) imaging at least a portion of the sample, and / or (b) Using optical imaging techniques, and / or (c) Using fluorescence imaging techniques, and / or (d) Multicolor fluorescence imaging techniques, and / or (e) Super-resolution fluorescence imaging technology.

[0062] The sequential demasking process can be applied in two different ways (see Figure 1): A) After in situ hybridization and before immunofluorescence staining. B) Before in situ hybridization or immunofluorescence staining.

[0063] Generally, a two-step demasking method is performed as follows: 1) Incubate the fixed tissue, which has been analyzed for in situ hybridization or has been newly prepared, with buffer A at 50–75°C for 0.5–2 hours. The preferred conditions are 70°C for 0.5 hours. 2) Allow the tissue to stand, transfer it to buffer B, and incubate at 50-75°C for 0.5-2 hours. The preferred conditions are 70°C for 0.5 hours. 3) Allow the tissue to stand, and then detect biomolecules using immunohistochemical staining or in situ hybridization.

[0064] In particular, the methods of this disclosure may be used to identify regions of interest, especially within organs, tissues, or single cells.

[0065] In several advantageous embodiments, the methods of the present disclosure may be used for image analysis. In this context, the methods of the present disclosure may be used to develop cell segmentation algorithms using immunofluorescence imaging. The methods of the present disclosure may be used to assist in the segmentation of cells in a sample, particularly in tissues. Cell segmentation is a critical step because errors in identifying cells and their boundaries directly affect the ability to accurately quantify protein expression levels in these cells. For example, information on protein levels in single cells may also be used to answer questions about the cell type assemblage in a sample, the spatial distribution of these types, and the interactions between different cells and cell types in tissues.

[0066] A unique advantage of spatial proteomics, unlike conventional methods, is that it does not require the prior selection of specific markers. Since these studies profile dozens of proteins, markers can be selected after the experiment. Furthermore, there is no need to select a single marker. Even if a single marker does not function for all cells, a combination of such markers may be useful for segmenting all cells.

[0067] Furthermore, the methods of this disclosure provide spatial information regarding the location of the profiled cells, thereby enabling the analysis of intercellular signaling and cell type organization. By using the methods of this disclosure to detect analytes such as biomolecules in fixed biological samples, up to three analytes such as proteins on the cell surface can be detected, thereby visualizing cell boundaries. The analytes may be bound by antibodies, antibody fragments, aptamers, or nucleic acids that specifically interact with and bind to the analyte, and antibody fragments in particular are selected from the group consisting of Fab, scFv, single domain, bis scFv, Fab2, Fab3, minibody, diabody, triplebody, tetrabody, and tandem antibodies (tandab).

[0068] In particular, the analytes detected may include sodium-potassium ATPase, plasma membrane calcium ATPase (PMCA), cadherin, CD98, caveolae, HER2, β-catenin, CAH9, E-cadherin, c-MYC, vimentin, etc. The selection of binding elements, such as antibodies, varies based on the cell type being detected. Each tissue has a different cell type composition. By visualizing cell boundaries, users can segment cells visually or via computational tools.

[0069] In some further advantageous embodiments, the methods of the present disclosure may be used to establish AI-based image analysis algorithms for identifying diseases using immunofluorescence imaging. Furthermore, in advantageous embodiments, the methods of the present disclosure may be used for feature extraction such as nuclear morphology, signal homogeneity, signal intensity, and / or center of gravity. For example, cell morphology often reflects its tissue-specific function or state. Cells undergo morphological changes to acquire motility in response to various stimuli, which may be part of normal physiology and development or due to pathological damage. In some cancers, epithelial cells lose cell-cell adhesion and exhibit protruding and exploratory morphology as they transition from a static phenotype to a migratory phenotype. Recognizing such abnormal cell morphology can help in the proper identification of malignant tumors (as distinguished from benign tumors).

[0070] A further application of the methods disclosed herein is to determine the expression of target proteins and genes through semi-quantitative approaches. For example, semi-quantitative immunohistochemistry (IHC) is a powerful method for investigating protein expression and localization within tissues.

[0071] In some further advantageous embodiments, the methods of the present disclosure may be used for diagnostic and prognostic applications. In particular, the methods of the present disclosure may be used to determine the distribution of a target protein in healthy and diseased organs, tissues, and / or cells. Furthermore, the methods of the present disclosure may be used to predict the prognosis of a disease using disease-related prognostic biomarkers and / or predictive biomarkers. A variety of factors influence a patient's clinical outcome, including intrinsic characteristics of the patient, disease, or medical condition, and the effects of any treatments the patient receives. Some of these intrinsic characteristics may be reflected as prognostic biomarkers, i.e., biomarkers used to identify clinical events, the likelihood of disease recurrence or progression in a patient with a particular disease or medical condition of interest, while others may be reflected as predictive biomarkers, i.e., biomarkers used to identify individuals who are more likely to be favorably or unfavorably affected by exposure to a medical product or environmental factor than similar individuals who do not possess the biomarkers.

[0072] In some further advantageous embodiments, the methods of the present disclosure may be used to provide disease diagnoses, for example, tumors of unknown primary origin, neurodegenerative diseases, muscle diseases, traumatic brain injury, and infectious diseases. In particular, the methods of the present disclosure are used to predict therapeutic response. A further application of the methods of the present disclosure is disease subclassification. Disease classification is crucial in compiling statistics on the causes of disease (incidence rates) and causes of death (mortality rates).

[0073] Furthermore, the methods of this disclosure may be used to examine gene expression in normal and pathological tissues and to study gene regulation.

[0074] In some further advantageous embodiments, the methods of the present disclosure may be used for morphological studies. In particular, the methods of the present disclosure may be used for the recognition of tissue structures and / or cell type classification. In this context, cell type is a classification used to identify cells that share morphological or phenotypic characteristics. Multicellular organisms may contain a wide variety of cell types, such as muscle cells and skin cells, which differ in both appearance and function but have identical genomic sequences. Cells may belong to different cell types even if they have the same genotype, due to the differential regulation of the genes they contain. Classification of specific cell types is often performed using a microscope (e.g., derived from a group of differentiation antigens commonly used for this purpose in immunology).

[0075] In some further advantageous embodiments, the methods of the present disclosure may be used in multi-omics research. Multi-omics is a biological analysis approach that uses datasets consisting of multiple “omes,” such as genome, proteome, transcriptome, epigenome, metabolome, and microbiome. By combining these “omes,” scientists can analyze complex biological big data to find novel associations between biological entities, identify relevant biomarkers, and construct sophisticated markers of disease and physiology. In this way, multi-omics integrates diverse omics data to find consistent and corresponding geno-pheno-envirotype relationships or associations. In particular, the methods of the present disclosure are used to study quantitative correlations and spatial colocalization between ISH and ICH. As previously mentioned, immunohistochemistry (IHC) is an extremely useful tool for detecting, localizing, and quantifying antigens in preserved tissues for research and diagnostic purposes. In situ hybridization (ISH) is a unique molecular analysis method that provides precise microscopic localization of analytes such as proteins, DNA, mRNA, and nucleic acids such as microRNA in metaphase chromosome specimens and cell and tissue preparations. In particular, the method disclosed herein is used for cell type classification based on such multi-omics data. In yet another embodiment, the method disclosed herein is used to study epigenetic modifications and chromatin structure. In particular, the method disclosed herein is used to identify spatial distribution and cell-specific ISH signals (cell segmentation).

[0076] In some further advantageous embodiments, the methods of the present disclosure may be used to determine the correlation between genetic abnormalities and / or copy number variations (CNVs) and corresponding proteins, where CNVs are a phenomenon in which sections of the genome are repeated and the number of repeats in the genome differs from one individual to another.

[0077] Experimental data Frozen sections of mouse kidney tissue were sectioned to a thickness of 10 μm using a cryostat (Leica Biosystems, CM1520). The tissue was dehydrated, rehydrated, fixed with 4% paraformaldehyde for 30 minutes, and processed using a two-step demasking method. Specifically, the tissue was first incubated in pH 6 buffer at 70°C for 30 minutes. Next, the buffer was replaced with pH 9 buffer, and the tissue was incubated at 70°C for another 30 minutes. Subsequently, the tissue was probed for lysozyme, MDM2, and p21. Rabbit primary antibody against lysozyme (BioLegend, #860001), as well as mouse primary antibodies against MDM2 (Santa Cruz, sc-965) and p21 (Origene, AM09100PU-T), were incubated on the tissue. After incubation of the primary antibody, Alexa Flour 647 (Abcam, ab150083) conjugated to goat anti-rabbit IgG was added. Images were acquired using a ZEISS Celldiscoverer 7 with an Apochromat 5X / 0.35 objective lens and a 2X tube lens (see Figure 2).

[0078] Frozen sections of tissue derived from a colorectal cancer xenograft model were sectioned to a thickness of 10 μm using a cryostat (Leica Biosystems, CM1520). The tissue was dehydrated, rehydrated, and fixed with 4% paraformaldehyde. Molecular cartography® was performed to detect RNA transcripts of target genes. After RNA transcript detection, the tissue was treated with a two-step demasking method. Specifically, the tissue was first incubated in pH 6 buffer at 70°C for 30 minutes. Next, the buffer was replaced with pH 9 buffer, and the tissue was incubated at 70°C for another 30 minutes. Subsequently, the tissue was probed for CTNNB1, CD34, and vimentin. Rabbit primary antibody against CTNNB1 (Cell Signaling Technology, 8480S), mouse primary antibody against CD34 (Invitrogen, #14-0341-82), and chicken primary antibody against vimentin (BioLegend, #919101) were incubated on tissue. After incubation of the primary antibodies, goat anti-rat IgG conjugated with Alexa Fluor® 594 (Abcam, ab150168), goat anti-rabbit IgG conjugated with Alexa Flour® 546 (Invitrogen, A11010), and goat anti-chicken IgY conjugated with Alexa Fluor® 488 were added. Images were acquired using a ZEISS Celldiscoverer 7 with an Apochromat 50X / 1.2 objective lens and a 0.5X tube lens (see Figure 3).

Claims

1. A method for antigen retrieval from a fixed sample, comprising the following steps: a) Incubating the fixed sample in the first antigen retrieval solution at a temperature of 50 to 75°C, b) Transferring the fixed sample to the second antigen retrieval solution, c) Incubating the fixed sample in the second antigen retrieval solution at a temperature of approximately 50 to 75°C. Includes, Here, the first antigen retrieval solution contains a buffer having a pH range of about 5 to 7, and the second antigen retrieval solution contains a buffer having a pH range of about 8 to 10, or The first antigen retrieval solution comprises a buffer having a pH range of approximately 8 to 10, and the second antigen retrieval solution comprises a buffer having a pH range of approximately 5 to 7. method.

2. The method according to claim 1, wherein the fixed sample is a biological sample, preferably comprising biological tissue derived from a human, animal, or plant, and more preferably comprising biological cells and / or extracts and / or a portion of cells.

3. The method according to any one of claims 1 or 2, wherein the cells are prokaryotic or eukaryotic cells, animal cells, plant cells, and in particular cells derived from vertebrate cells, in particular mammalian cells, and in particular human cells.

4. The method according to any one of claims 1 to 3, wherein the fixed sample is a frozen fixed tissue sample, and more particularly a frozen section of tissue.

5. The method according to any one of claims 1 to 4, wherein the sample is fixed in a crosslinking fixing agent.

6. The method according to any one of claims 1 to 5, wherein the sample is fixed in a coagulation-fixing agent such as alcohol and acetone.

7. The method according to any one of claims 1 to 6, wherein the sample is fixed in formaldehyde and embedded in paraffin (FFPE), and is in particular a frozen section of a formaldehyde-fixed biological sample.

8. The method according to any one of claims 1 to 7, wherein the sample is a cryopreserved tissue sample and is post-fixed with a coagulation agent such as alcohol or acetone, and / or a crosslinking agent such as formaldehyde.

9. The method according to any one of claims 1 to 8, wherein the fixed sample is incubated for at least 0.5 hours, particularly about 0.5 to 2 hours, in step a).

10. The method according to any one of claims 1 to 9, wherein the fixed tissue sample is incubated in step b) for at least 0.5 hours, particularly about 0.5 to 2 hours.

11. The method according to any one of claims 1 to 10, wherein the fixed tissue sample is incubated at a temperature of about 0.5 and about 70°C in step a).

12. The method according to any one of claims 1 to 11, wherein the fixed tissue sample is incubated at a temperature of about 0.5 and about 70°C in step b).

13. The method according to any one of claims 1 to 12, wherein the buffer having a pH range of approximately 5 to 7 contains citric acid, and the buffer having a pH range of approximately 8 to 10 contains tris(hydroxymethyl)methylamine (TRIS).

14. The method according to any one of claims 1 to 13, wherein the buffer having a pH range of about 5 to about 7 comprises citric acid, potassium dihydrogen phosphate, boric acid, diethylbarbituric acid, piperazine-N,N'-bis(2-ethanesulfonic acid), dimethylarsinic acid, 2-(N-morpholino)ethanesulfonic acid, or a combination thereof.

15. The method according to any one of claims 1 to 14, wherein the buffer solution having a pH range of approximately 5 to approximately 7 contains citric acid.

16. The method according to any one of claims 1 to 15, wherein the buffer solution having a pH range of approximately 8 to 10 comprises tris(hydroxymethyl)methylamine (TRIS), 2-(N-morpholino)ethanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-tris(hydroxymethyl)methylglycine (Tricine), 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), or a combination thereof.

17. The method according to any one of claims 1 to 16, wherein the buffer having a pH range of approximately 8 to 10 comprises TRIS and / or boric acid.

18. The method according to one or more prior claims, wherein the fixed tissue sample is a section of an organ or tissue, body fluid, tissue, or a microarray.

19. The method according to one or more prior claims, wherein the incubation step with the first antigen retrieval solution and the second antigen retrieval solution includes incubation in a heating device.

20. The method according to claim 19, wherein the heating device is a pressure cooker, oven, stove, autoclave, water bath, hot plate, microwave, steam heating, or a combination thereof.

21. The method according to one or more prior claims, further comprising the step of immunostaining of an antigen.

22. The method according to claim 21, wherein the immunostaining comprises sequential immunoperoxidase labeling and erasure.

23. The method according to one or more prior claims, wherein the fixed tissue sample undergoes in-situ hybridization analysis.

24. The method according to one or more prior claims, wherein the fixed tissue sample is subjected to FISH or CISH analysis.

25. The method according to one or more prior claims, wherein the fixed tissue sample is subjected to spatial biological analysis such as spatial transcriptomics and / or spatial proteomics.

26. The method according to one or more prior claims, wherein the fixed tissue sample is analyzed by in-situ hybridization before antigen retrieval, and the fixed tissue sample is analyzed by immunohistochemical staining of the antigen after antigen retrieval.

27. The method according to one or more prior claims, wherein, after antigen retrieval, the fixed tissue sample is analyzed by in-situ hybridization and / or immunohistochemical staining of the antigen.

28. The method according to any one of claims 1 to 27, wherein the analyte is immobilized in a permeabilized sample such as a sample containing cells.

29. The method according to any one of claims 1 to 28, wherein the handling of one or more of the samples is automated.

30. The method according to any one of claims 1 to 5, wherein the antigen is a nucleic acid, preferably DNA, PNA, or RNA, particularly mRNA.

31. The method according to any one of claims 1 to 5, wherein the antigen is a peptide, polypeptide, or protein.

32. The method according to any one of claims 1 to 31, wherein the entire process is automated, in particular by using a robotic system.

33. The method according to any one of claims 1 to 32, wherein the entire process is carried out within a fluid system.

34. The aforementioned antigen is as follows: (a) Imaging at least a portion of the sample, and / or (b) Using optical imaging techniques, and / or (c) Using fluorescence imaging techniques, and / or (d) Multicolor fluorescence imaging techniques, and / or (e) Super-resolution fluorescence imaging technology The method according to any one of claims 1 to 33, as detected by...

35. Identifying areas of interest, image analysis, development of cell segmentation algorithms using immunofluorescence imaging, assistance in segmenting cells in samples, construction of AI-based image analysis algorithms for identifying defects using immunofluorescence imaging, feature extraction such as nuclear morphology, signal homogeneity, signal intensity, and / or centroid, semi-quantitative determination of the expression of target proteins and genes, diagnostic and prognostic applications, determination of the distribution of target proteins in organs, tissues, and / or cells in health and disease, and prediction of disease prognosis using disease-related prognostic and / or predictive biomarkers, e.g., tumors of unknown primary origin, neurodegenerative diseases, muscle diseases. The method according to any of the prior claims for use in applications selected from the group consisting of providing disease diagnoses such as traumatic brain injury and infectious diseases, predicting therapeutic responses, subclassifying diseases, examining gene expression and gene regulation in normal and pathological tissues, morphological studies, recognition of tissue structure and / or cell type classification, multi-omics studies, studying quantitative correlations and spatial colocalization between ISH and IHC, studying epigenetic modifications and chromatin structure, identifying spatial distribution and cell-specific ISH signals (cell segmentation), and determining the correlation between genetic abnormalities and / or copy number variations (CNVs) and corresponding proteins.

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