Immunostaining reagent for MHC-II expression detection of gastric cancer biopsy sample

Abstract

The invention discloses an immunostaining reagent for MHC-II expression detection of a gastric cancer biopsy sample, and belongs to the field of bioengineering. The invention establishes a preparation method of an immunohistochemical staining section. The preparation method specifically comprises the following steps: step 1, early-stage treatment: baking a tissue section, dewaxing, rehydrating, subsequently adding an antigen solution for repairing, adding an endogenous enzyme inhibitor for incubation, adding a sealing agent for sealing and the like; step 2, antibody incubation: adding a first antibody and a second antibody for incubation; and step 3, post-treatment: dyeing, re-dyeing, bluing, sealing and the like. A mixed buffer solution is used as an endogenous enzyme inhibitor, so that the background staining intensity is effectively reduced, and the staining sensitivity and specificity are improved; meanwhile, a staining result interpretation standard is established, it is proved that MHC-II expression is remarkably related to pathological remission of the gastric cancer patient, and an evaluation method is provided for pathological remission of the gastric cancer patient.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and in particular relates to an immunostaining reagent for detecting MHC-II expression in gastric cancer biopsy samples. Background Art

[0002] Gastric and gastroesophageal junction (G / GEJ) cancer ranks fifth among newly diagnosed malignancies and fourth among cancer-related deaths worldwide. Despite recent advances in multidisciplinary or multimodal treatment, the prognosis for patients with locally advanced disease remains poor, with a median overall survival (OS) of only 34.4 months and a 5-year OS rate of only 38.7%. According to the Chinese Society of Clinical Oncology (CSCO) guidelines, perioperative therapy combined with D2 gastrectomy has become the standard treatment for locally advanced G / GEJ cancer (cT3-4aN+M0). However, the optimal perioperative treatment regimen and sequence remain unclear. Although perioperative treatment has achieved high R0 resection rates exceeding 90%, optimizing treatment options is necessary to improve clinical outcomes for patients with locally advanced G / GEJ cancer.

[0003] Screening patients with G / GEJ cancer to identify those who may benefit from perioperative immunotherapy is crucial. Currently, biomarkers associated with immunotherapy efficacy primarily include PD-L1 expression, microsatellite instability (MSI) status, Epstein-Barr virus (EBV) infection status, and tumor-infiltrating lymphocytes (TILs). Theoretically, cancer patients with high PD-L1 expression, microsatellite instability-high (MSI-H) status, or EBV infection are more likely to exhibit enhanced responsiveness to immunotherapy. However, other patients may not fully respond to immunotherapy. Therefore, further exploration of increasingly precise biomarkers is crucial to identify the most suitable candidates for immunotherapy and improve its efficacy.

[0004] Major histocompatibility complex (MHC) molecules, also known as human leukocyte antigen (HLA) molecules, are currently being studied. Studies have shown that the immune system recognizes tumor cells through major histocompatibility complexes (MHCs). MHCs are cell surface receptors that bind foreign peptides and present them to T lymphocytes. MHC class II molecules, encoded by polymorphic MHC genes, consist of a non-covalent complex of α and β chains. Helper T lymphocytes bind antigenic peptides presented by MHC class II molecules. MHC class II molecules bind antigenic peptides of 13-18 amino acids. HLA-DM and -DO molecules accumulate in endosomal / lysosomal compartments and on the surface of B cells, regulating the binding of foreign peptides to class II molecules (HLA-DR) by maintaining a conformation that favors peptide exchange. These molecules are expressed in various tumor tissues and are crucial for antigen presentation by CD4+ T lymphocytes, whose role in anti-tumor immunity is increasingly recognized. MHC-II is associated with higher numbers of CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs), a lack of lymphovascular invasion, increased formation of tertiary lymphoid structures, upregulation of genes involved in IFN-γ pathway activation (including the CD274 gene encoding PD-L1), and higher levels of IFNγ, IL2, and IL12 mRNA (Th1 cytokines). Overall, increased expression of MHC-II or related pathway components in tumor cells is associated with better prognosis and enhanced anti-tumor immunity, thus playing an important role in immunotherapy.

[0005] MHC-II expression can be detected through immunohistochemical staining, but it is not yet a routine indicator in clinical pathology. Therefore, there are no unified standards and methods for its preparation, staining, and interpretation, and its screening value still requires further clinical and basic validation. Therefore, the existing technology still needs a technology that can obtain high-quality and accurate MHC-II immunohistochemical staining sections in a relatively short time, as well as a complete and accurate standard for interpreting pathological sections. Summary of the Invention

[0006] The purpose of the present invention is to provide an immunostaining reagent for detecting MHC-II expression in gastric cancer biopsy samples, so as to solve the problems of MHC-II expression immunohistochemical staining technology detection, preparation, staining and interpretation without unified standards and methods.

[0007] In order to solve the above technical problems, the present invention specifically provides the following technical solutions: The present invention discloses a mixed buffer for use in preparing endogenous enzyme inhibitors. The mixed buffer contains a hydrazine derivative and amino-polyethylene glycol-galactose, with the mass ratio of the hydrazine derivative to the amino-polyethylene glycol-galactose in the mixed buffer being 1:0.2-1. The mixed buffer of the hydrazine derivative and amino-polyethylene glycol-galactose can effectively inhibit endogenous enzymes, preventing the endogenous enzymes from reacting with a matrix solution and causing false positives, thereby significantly reducing nonspecific background and improving the sensitivity and specificity of staining.

[0008] Preferably, the mixed buffer further contains a phosphate buffer, and the amount of the hydrazine derivative in the phosphate buffer is 0.045-0.06 g / ml.

[0009] Preferably, the hydrazine derivative is prepared from 1-hydroxy-2-naphthoic acid hydrazide and acetic anhydride, and the mass ratio of the amount of 1-hydroxy-2-naphthoic acid hydrazide used to the amount of acetic anhydride used is 1:0.45-0.7.

[0010] The invention discloses an immunostaining reagent for detecting MHC-II expression in gastric cancer biopsy samples. The immunostaining reagent comprises at least an endogenous enzyme inhibitor. The endogenous enzyme inhibitor comprises a mixed buffer solution containing a hydrazine derivative and amino-polyethylene glycol-galactose. The mass ratio of the hydrazine derivative to the amino-polyethylene glycol-galactose in the mixed buffer solution is 1:0.2-1.

[0011] Preferably, the immunostaining reagent further comprises a dewaxing and hydrating treatment solution, and the dewaxing and hydrating treatment solution is xylene, 95-100% alcohol, 90-95% alcohol and 75-85% alcohol.

[0012] Preferably, the immunostaining reagent further comprises an antigen repair solution, which is obtained by mixing citric acid and sodium citrate in water, wherein the mass ratio of citric acid to sodium citrate is 1:6-9, and the amount of citric acid added to water is 0.0003-0.0005 g / ml.

[0013] Preferably, the immunostaining reagent further comprises a blocking agent, and the blocking agent is goat serum.

[0014] Preferably, the immunostaining reagent further comprises an enhancing solution.

[0015] Preferably, the immunostaining reagent further comprises a primary antibody, which is a diluted MHC Class II recombinant rabbit monoclonal antibody, and the diluted MHC Class II recombinant rabbit monoclonal antibody is prepared by mixing phosphate buffer and MHC Class II recombinant rabbit monoclonal antibody in a volume ratio of 1:0.002-0.015.

[0016] The present invention also discloses an immunoassay kit based on MHC-II expression, comprising the above-mentioned immunostaining reagent.

[0017] The present invention discloses a method for preparing immunohistochemically stained sections, comprising: Step 1: Preliminary treatment, adding a dewaxing and hydrating treatment solution to the tissue section for treatment, then immersing it in an antigen retrieval solution for repair, then adding an endogenous enzyme inhibitor for incubation, and finally adding a blocking agent for blocking to obtain a treated tissue section; the endogenous enzyme inhibitor is at least one of 1-3% hydrogen peroxide and a mixed buffer, and the mixed buffer is obtained by dissolving a hydrazine derivative and amino-polyethylene glycol-galactose in a phosphate buffer.

[0018] Step 2: Antibody incubation: adding the first antibody to the treated tissue section obtained in step 1 for incubation, then immersing it in an enhancement solution and letting it stand, and then adding the second antibody for incubation. After the incubation is completed, a tissue section complex is obtained; Step 3: Post-processing: the tissue section complex obtained in step 2 is sequentially added with a color developer, a counterstain, and an anti-blue agent for staining, and then dehydrated and sealed to obtain an immunohistochemical staining section.

[0019] Preferably, in step 1, the amount of the hydrazine derivative in the phosphate buffer is 0.045-0.06 g / ml, and the mass ratio of the amount of the hydrazine derivative to the amount of amino-polyethylene glycol-galactose is 1:0.2-1.

[0020] Preferably, the dewaxing and hydration treatment liquid in step 1 includes xylene, 95-100% alcohol, 90-95% alcohol and 75-85% alcohol; the xylene treatment time is 3-10 minutes, and the 95-100% alcohol, 90-95% alcohol and 75-85% alcohol are treated in sequence for 3-10 minutes.

[0021] Preferably, in step 1, the antigen retrieval solution is prepared by mixing citric acid and sodium citrate in water, the mass ratio of citric acid to sodium citrate is 1:6-9, and the amount of citric acid added to water is 0.0003-0.0005 g / ml; the blocking agent is sheep serum.

[0022] Preferably, the amount of dewaxing and hydrating solution, antigen retrieval solution, endogenous enzyme inhibitor and blocking agent used in step 1 is enough to completely immerse the tissue section.

[0023] Preferably, in step 1, the antigen retrieval time is 60-120 seconds; the endogenous enzyme inhibitor incubation time is 5-15 minutes; and the blocking agent blocking time is 5-15 minutes.

[0024] Preferably, in step 2, the first antibody is a diluted MHC Class II recombinant rabbit monoclonal antibody, and the second antibody is an enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody; the diluted MHC Class II recombinant rabbit monoclonal antibody is prepared by mixing phosphate buffer and MHC Class II recombinant rabbit monoclonal antibody in a volume ratio of 1:0.002-0.015; the amount of the first antibody, enhancement solution, and second antibody used is sufficient to completely immerse the tissue section.

[0025] Preferably, in step 2, the first antibody is added and the incubation temperature is 2-6°C, and the incubation time is 6-10h; after adding the enhancement solution, the standing temperature is 23-27°C, and the standing time is 15-30min; the second antibody is added and the incubation temperature is 23-27°C, and the incubation time is 15-30min. Preferably, in step 3, the color developer is DAB color developing solution, the counterstain is histochemical hematoxylin, and the anti-blueing agent is prepared by adding sodium bicarbonate to water and mixing, and the amount of sodium bicarbonate added to water is 0.001-0.003 g / ml; the amount of color developer, counterstain and anti-blueing agent used is to completely immerse the tissue section complex.

[0026] Preferably, after the color developer is added in step three, the incubation temperature is 23-27°C and the incubation time is 1-5 minutes; the immersion time after the counterstain is added is 1-5 minutes; the immersion time after the anti-blue solution is added is 1-5 minutes; the solvents used in the dehydration process are 95-100% alcohol, 90-95% alcohol and 75-85% alcohol, and the treatment is carried out in sequence for 1-5 minutes.

[0027] The invention discloses a gastric cancer biopsy sample immunostaining reagent based on MHC-II expression, comprising: a pre-treatment reagent, an antibody incubation reagent and a post-treatment reagent.

[0028] Preferably, the pre-treatment reagents include dewaxing and hydration treatment solution, antigen repair solution, inhibitor and blocking agent; the antibody incubation reagents include primary antibody, enhancement solution and secondary antibody; the post-treatment reagents include color developer, counterstain, anti-blue agent and dehydration solvent.

[0029] The present invention discloses a method for preparing immunohistochemically stained sections, comprising: Step 1: Preliminary treatment: slice the cancer tissue wax block to obtain tissue sections, process the tissue sections, and then immerse them in antigen retrieval solution for high-pressure repair treatment for 60-120 seconds. Then, draw the tissue size, add endogenous enzyme inhibitors and incubate for 5-15 minutes, and then add blocking agent for blocking for 5-15 minutes to obtain processed tissue sections.

[0030] Step 2: Antibody incubation: Add the primary antibody to the treated tissue section obtained in step 1 and incubate at 2-6°C for 6-10 hours. Then, immerse the section in an enhancement solution at 23-27°C for 15-30 minutes. Then, add the secondary antibody to the treated tissue section and incubate at 23-27°C for 15-30 minutes. After the reaction is complete, a tissue section complex is obtained.

[0031] Step 3: Post-processing: Add color developing solution to the tissue section complex obtained in step 2 under light-proof conditions, incubate at 23-27°C for 1-5 minutes, immerse in counterstaining solution for 1-5 minutes, and then immerse in anti-blue solution for 1-5 minutes. Finally, dehydrate the tissue section complex, dry it, and seal it with a sealing agent.

[0032] Preferably, in the above technical solution, the mixed buffer used in step 1 is obtained by dissolving a hydrazine derivative and amino-polyethylene glycol-galactose in a phosphate buffer, the amount of the hydrazine derivative in the phosphate buffer is 0.045-0.06 g / ml, and the mass ratio of the amount of the hydrazine derivative used to the amount of amino-polyethylene glycol-galactose used is 1:0.2-1.

[0033] Preferably, in the above technical solution, the cancer tissue wax block is a biopsy tissue wax block of a patient with a first-time diagnosis of gastric and gastroesophageal junction tumor.

[0034] Preferably, in the above technical solution, the tissue section processing in step 1 is specifically as follows: baking the tissue sections at 60-75°C for 15-45 minutes, then immersing the tissue sections in xylene for 3-10 minutes, repeating the immersion three times, and after the immersion, taking out the tissue sections and placing them in 95-100% alcohol, 90-95% alcohol, 75-85% alcohol and water respectively, and then immersing them for 3-10 minutes each.

[0035] Preferably, in the above technical solution, the antigen retrieval solution used in step 1 is obtained by mixing citric acid and sodium citrate in water, the mass ratio of citric acid to sodium citrate is 1:6-9, and the amount of citric acid added to water is 0.0003-0.0005 g / ml.

[0036] Preferably, in the above technical solution, the endogenous enzyme inhibitor used in step 1 is at least one of 1-3% hydrogen peroxide and a mixed buffer.

[0037] Preferably, in the above technical solution, the blocking agent used in step 1 is sheep serum.

[0038] Preferably, in the above technical solution, the amount of solvent, antigen retrieval solution, endogenous enzyme inhibitor and blocking agent used in step 1 is enough to completely immerse the tissue section.

[0039] Preferably, in the above technical solution, the first antibody used in step 2 is a diluted MHC Class II recombinant rabbit monoclonal antibody, and the diluted MHC Class II recombinant rabbit monoclonal antibody is prepared by mixing phosphate buffer and MHC Class II recombinant rabbit monoclonal antibody in a volume ratio of 1:0.002-0.015.

[0040] Preferably, in the above technical solution, the second antibody used in step 2 is an enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody.

[0041] Preferably, in the above technical solution, the amount of the first antibody, the second antibody and the enhancement solution used in step 2 is enough to completely immerse the tissue section.

[0042] Preferably, in the above technical solution, the color developing solution used in step 3 is DAB color developing solution, and the counterstaining solution is histochemical hematoxylin.

[0043] Preferably, in the above technical solution, the anti-blue solution used in step 3 is obtained by adding sodium bicarbonate to water and mixing, and the amount of sodium bicarbonate added to the water is 0.001-0.003 g / ml.

[0044] Preferably, in the above technical solution, the amount of the color developing solution, counterstaining solution and anti-blueing solution used in step 3 is enough to completely immerse the tissue section complex.

[0045] Preferably, in the above technical solution, the solvents used in the dehydration process in step 3 are 95-100% alcohol, 90-95% alcohol and 75-85% alcohol, which are treated in sequence for 1-5 minutes.

[0046] Preferably, in the above technical solution, the slices need to be washed and dried after each treatment, that is, the slices are completely immersed in phosphate buffer for 3-10 minutes and dried. The slices include tissue slices, processed tissue slices and tissue slice complexes.

[0047] Preferably, the present invention discloses a method for preparing a hydrazine derivative, specifically: 1-Hydroxy-2-naphthoic acid is added to methanol, followed by the dropwise addition of concentrated sulfuric acid. The mixture is refluxed under nitrogen for 10-15 hours, followed by a 10-15 hour stand at 2-6°C to yield methyl 1-hydroxy-2-naphthoate. Methyl 1-hydroxy-2-naphthoate and NH₂NH₂·H₂O are then added to methanol, refluxed at 85-97°C for 1-5 hours, cooled to 23-27°C, and then allowed to stand at 2-6°C for 10-15 hours to yield 1-hydroxy-2-naphthoic acid hydrazide. Finally, 1-hydroxy-2-naphthoic acid hydrazide is added to a mixed solution of acetic anhydride and CHCl₃ at 0-4°C, heated to 23-27°C, reacted for 20-28 hours, and then allowed to stand at 2-6°C for 10-15 hours to yield a hydrazine derivative.

[0048] Preferably, in the preparation of the hydrazine derivative, the amount of 1-hydroxy-2-naphthoic acid added to methanol is 0.1-0.15 g / ml.

[0049] Preferably, in the preparation of the hydrazine derivative, the mass ratio of the amount of concentrated sulfuric acid used to the amount of 1-hydroxy-2-naphthoic acid used is 1:0.25-0.75.

[0050] Preferably, in the preparation of the hydrazine derivative, the mass ratio of the amount of 1-hydroxy-2-naphthoic acid methyl ester used to the amount of NH2NH2·H2O used is 1:2-3.5.

[0051] Preferably, in the preparation of the hydrazine derivative, the amount of methyl 1-hydroxy-2-naphthoate in methanol is 0.075-0.1 g / ml.

[0052] Preferably, in the preparation of the hydrazine derivative, the mass ratio of the amount of 1-hydroxy-2-naphthoic acid hydrazide used to the amount of acetic anhydride used is 1:0.45-0.7, and the amount of acetic anhydride in the mixed solution of acetic anhydride and CHCl3 is 0.01-0.025g / ml.

[0053] Preferably, the immunohistochemical staining method of the present invention, in addition to using a hydrazine derivative and an amino-polyethylene glycol-galactose mixed buffer, may further include a glycerol polyoxyethylene ether cocoate buffer to replace the sheep serum. The glycerol polyoxyethylene ether cocoate buffer effectively reduces and eliminates nonspecific sites, preventing nonspecific interactions between antibodies or detection reagents and these nonspecific sites, thereby improving the specificity of the staining, reducing background staining, and allowing the antibody to bind only to the target antigen, thereby improving the sensitivity and specificity of the staining.

[0054] Preferably, the mixed buffer and the glyceryl polyoxyethylene ether cocoate buffer are used in amounts sufficient to completely immerse the tissue sections.

[0055] Compared with the prior art, the present invention has the following beneficial effects: The present invention establishes a method for preparing sections for immunohistochemical staining, specifically comprising step 1: pre-treatment, including baking, dewaxing, and rehydrating the tissue sections, followed by repair with an antigen solution, incubation with endogenous enzyme inhibitors, and blocking with a blocking agent; step 2: antibody incubation, including incubation with the first and second antibodies; and step 3: post-treatment, including staining, counterstaining, anti-blueing, and sealing. Using a mixed buffer as an endogenous enzyme inhibitor effectively reduces background staining intensity and improves staining sensitivity and specificity. A standard for interpreting staining results was also established, confirming that MHC-II expression is significantly correlated with pathological remission in gastric cancer patients, providing an evaluation method for pathological remission in gastric cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0057] Figure 1 Schematic diagram of the standard for interpreting staining results; Figure 2 This is a diagram of MHC II expression efficiency. DETAILED DESCRIPTION

[0058] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0059] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is intended only to make the content of this application easier to understand and does not limit the scope of protection of this application. At the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following detailed description of this application will be made with reference to the accompanying drawings and in conjunction with the embodiments.

[0060] Example 1: A method for preparing sections for immunohistochemical staining: Step 1: Preliminary processing: Biopsy tissue blocks from patients with first-time gastric and gastroesophageal junction cancers are sliced ​​and processed to obtain tissue sections. The sections are then baked at 72°C for 20 minutes and then immersed in xylene for 5 minutes. This immersion is repeated three times. After immersion, the sections are removed and immersed in 100% alcohol, 95% alcohol, 80% alcohol, and purified water for 3 minutes each. The sections are then removed and retrieval is performed in antigen retrieval solution at high pressure for 90 seconds. After retrieval, the sections are washed and immersed in phosphate buffered saline for 5 minutes. The sections are then dried, marked, and incubated in 3% hydrogen peroxide for 10 minutes. After incubation, the sections are washed and immersed in phosphate buffered saline for 5 minutes. The sections are then removed and blocked in goat serum for 10 minutes. After blocking, the processed tissue sections are obtained. The antigen retrieval solution is prepared by mixing citric acid and sodium citrate in water. The mass ratio of citric acid to sodium citrate is 1:7.5. The amount of citric acid added to water is 0.0004 g / ml. The amount of 3% hydrogen peroxide and sheep serum used is to completely immerse the tissue sections.

[0061] Step 2: Antibody incubation: Add diluted MHC Class II recombinant rabbit monoclonal antibody to the treated tissue sections and incubate at 4°C for 8 hours. After the reaction, wash the treated tissue sections and then immerse them in phosphate buffered saline for 5 minutes. Then, spin dry the treated tissue sections, immerse them in enhancement solution at 25°C for 20 minutes, wash them again, and immerse them in phosphate buffered saline for 5 minutes. After immersion, spin dry the treated tissue sections, add enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody to the treated tissue sections, incubate them at 25°C for 20 minutes, wash them, and immerse them in phosphate buffered saline for 5 minutes to obtain a tissue section complex. The amount of diluted MHC Class II recombinant rabbit monoclonal antibody and enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody used was sufficient to completely immerse the tissue sections. The diluted MHC Class II recombinant rabbit monoclonal antibody was prepared by mixing phosphate buffer and MHC Class II recombinant rabbit monoclonal antibody in a volume ratio of 1:0.01. The MHC Class II recombinant rabbit monoclonal antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd. with the catalog number ET1704-13. The enhancing solution and enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody were derived from the detection kit, which was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. with the catalog number PV-9001.

[0062] Step 3: Post-processing, add DAB colorimetric solution to the tissue section complex under light-proof conditions, incubate at 25°C for 3 minutes, wash with water and dry. Add hematoxylin staining solution and soak for 2 minutes, wash with water, and dry. Then immerse in anti-blue solution and soak for 2 minutes, wash with water. Finally, soak in 100% alcohol, 95% alcohol, 80% alcohol and pure water for 3 minutes each. Dry and seal with sealing agent. The anti-blue solution is obtained by adding sodium bicarbonate to water and mixing. The amount of sodium bicarbonate added to water is 0.002g / ml. The amount of DAB colorimetric solution, hematoxylin staining solution and anti-blue solution used is to completely immerse the tissue sections. Hematoxylin staining solution and sealing agent are purchased from Ningbo Tongsheng Biotechnology Co., Ltd.

[0063] Example 2: Preparation of hydrazine derivatives: 1-Hydroxy-2-naphthoic acid is added to methanol to obtain a mixture. Concentrated sulfuric acid is then added dropwise to the mixture, which is refluxed under nitrogen for 14 hours and then allowed to stand at 4°C for 12 hours to obtain 1-hydroxy-2-naphthoic acid methyl ester. Next, 1-hydroxy-2-naphthoic acid methyl ester and NH₂NH₂·H₂O are added to methanol, refluxed at 95°C for 3 hours, cooled to room temperature, and then allowed to stand at 4°C for 12 hours to obtain 1-hydroxy-2-naphthoic acid hydrazide. Finally, 1-hydroxy-2-naphthoic acid hydrazide is added to a mixed solution of acetic anhydride and CHCl₃ at 0°C, heated to 25°C, reacted for 24 hours, and then allowed to stand at 4°C for 12 hours to obtain a hydrazine derivative. The amount of 1-hydroxy-2-naphthoic acid added to methanol is 0.12 g / ml, the mass ratio of concentrated sulfuric acid to 1-hydroxy-2-naphthoic acid is 1:0.5, the mass ratio of 1-hydroxy-2-naphthoic acid methyl ester to NH2NH2·H2O is 1:2.5, the amount of 1-hydroxy-2-naphthoic acid methyl ester in methanol is 0.089 g / ml, the mass ratio of 1-hydroxy-2-naphthoic acid hydrazide to acetic anhydride is 1:0.59, and the amount of acetic anhydride in the mixed solution of acetic anhydride and CHCl3 is 0.019 g / ml.

[0064] A method for preparing immunohistochemically stained sections: The preparation of immunohistochemically stained sections in this embodiment is different from that in Example 1, except that 3% hydrogen peroxide is replaced with a mixed buffer solution, the mixed buffer solution is obtained by dissolving a hydrazine derivative and amino-polyethylene glycol-galactose in a phosphate buffer solution, the amount of the hydrazine derivative in the phosphate buffer solution is 0.054 g / ml, the mass ratio of the amount of the hydrazine derivative used to the amount of amino-polyethylene glycol-galactose used is 1:0.55, and the amount of the mixed buffer solution used is sufficient to completely immerse the tissue section. Other conditions and parameters are the same as in Example 1.

[0065] Example 3: The preparation of the hydrazine derivative is the same as in Example 2.

[0066] A method for preparing immunohistochemically stained sections: The preparation of immunohistochemically stained sections in this embodiment is different from that in Example 2, except that the mass ratio of the amount of hydrazine derivative used and the amount of amino-polyethylene glycol-galactose used is 1:0.8, and other conditions and parameters are the same as in Example 2.

[0067] Example 4: The preparation of the hydrazine derivative is the same as in Example 2.

[0068] A method for preparing immunohistochemically stained sections: The preparation of immunohistochemically stained sections in this embodiment is different from that in Example 2, except that the mass ratio of the amount of hydrazine derivative used and the amount of amino-polyethylene glycol-galactose used is 1:0.25, and other conditions and parameters are the same as in Example 2.

[0069] Example 5: The preparation of the hydrazine derivative is the same as in Example 2.

[0070] A method for preparing immunohistochemically stained sections: The preparation of immunohistochemically stained sections in this embodiment is compared with that in Example 2, except that sheep serum is replaced with glycerol polyoxyethylene ether cocoate buffer, and the amount of glycerol polyoxyethylene ether cocoate in phosphate buffer is 0.15 g / ml.

[0071] Example 6: The preparation of the hydrazine derivative is the same as in Example 2.

[0072] A method for preparing immunohistochemically stained sections: The preparation of immunohistochemically stained sections in this embodiment is compared with that in Example 2, except that sheep serum is replaced with glycerol polyoxyethylene ether cocoate buffer, and the amount of glycerol polyoxyethylene ether cocoate in phosphate buffer is 0.25 g / ml.

[0073] Comparative Example 1: A method for preparing immunohistochemically stained sections: The preparation of immunohistochemically stained sections in this example is different from that in Example 2 in that no hydrazine derivative is used in the mixed buffer solution, and other conditions and parameters are the same as in Example 2.

[0074] Comparative Example 2: The preparation of the hydrazine derivative is the same as in Example 2.

[0075] A method for preparing immunohistochemically stained sections: The preparation of immunohistochemically stained sections in this example is different from that in Example 2, except that amino-polyethylene glycol-galactose is not used in the mixed buffer solution, and other conditions and parameters are the same as in Example 2.

[0076] Experimental Example 1: Background staining intensity was evaluated on the immunohistochemical sections prepared in Examples 1-6 and Comparative Examples 1-2. "-": no staining; "±": weakly positive and very weak locally; "+": diffusely and weakly positive; "++": diffusely and moderately positive; "+++": diffusely and strongly positive.

[0077] Table 1 Results of background staining intensity evaluation of immunohistochemically stained sections

[0078] The results of background staining intensity are shown in Table 1. The background staining intensity of the immunohistochemically stained sections prepared by the immunohistochemical staining method of Example 2 is lower than that of Example 1, indicating that the use of the hydrazine derivative buffer and the amino-polyethylene glycol-galactose liquid effectively reduces the background staining intensity, thereby reducing interference with subsequent interpretation; the background staining intensity of the immunohistochemically stained sections prepared by the immunohistochemical staining method of Example 5 is lower than that of Example 2, indicating that the use of the hydrazine derivative buffer and the amino-polyethylene glycol-galactose liquid and the use of glycerol polyoxyethylene ether cocoate can further improve the accuracy and specificity of the staining and reduce the background staining intensity; the background staining intensity of the immunohistochemically stained sections prepared by the immunohistochemical staining methods of Comparative Examples 1 and 2 is higher than that of Example 2, indicating that the hydrazine derivative buffer and the amino-polyethylene glycol-galactose liquid need to work together, and the use of either the hydrazine derivative buffer or the amino-polyethylene glycol-galactose liquid alone has no significant effect on reducing the background staining intensity.

[0079] Experimental Example 2: The pathological response of gastric cancer was evaluated based on MHC II expression. First, biopsy tissue wax blocks from patients with first-time gastric and gastroesophageal junction tumors were used to obtain immunohistochemically stained sections according to the preparation method of immunohistochemically stained sections in Example 1. The staining results were interpreted and divided into negative and positive expression groups based on the interpretation criteria and the staining results. The result interpretation criteria were: 0: no staining or ≤10% of cancer cells showed incomplete and weak cell membrane staining; 1+: >10% of cancer cells showed incomplete and weak cell membrane staining; 2+: >10% of cancer cells showed weak to moderate intensity intact cell membrane staining, ≤10% of cancer cells showed strong and intact cell membrane staining; 3+: Strong, complete, and uniform cell membrane staining in >10% of cancer cells.

[0080] like Figure 1 As shown, an immunohistochemical score of 0 or 1+ indicates negative expression, and a score of 2+ or 3+ indicates positive expression.

[0081] Pathological response was then evaluated for both negative and positive expression in 235 gastric cancer patients undergoing radical resection and neoadjuvant therapy (chemotherapy plus immunotherapy) using a chi-square test. The pathological complete response rate (pCR) was defined as the absence of residual tumor cells detected under a light microscope (including lymph nodes, stage ypT0N0M0), while the major pathological response rate (mPR) was defined as a tumor regression grade (TRG) of 0 or 1. The criteria for pathological response are shown in Table 1.

[0082] Table 2 Tumor pathological response evaluation criteria

[0083] Evaluation results such as Figure 2 As shown in the results, MHC II expression levels were significantly correlated with pCR and mPR, indicating the potential importance of MHC II in the evaluation of pathological response in gastric cancer.

[0084] Experimental Example 3: For the sensitivity and specificity test, biopsy tissue wax blocks from 50 patients with gastric and gastroesophageal junction tumors (gastric cancer group) and tissue wax blocks from 50 patients with non-gastric and gastroesophageal junction tumors (non-gastric cancer group) were taken. Immunohistochemical staining sections were obtained according to the preparation method of immunohistochemical staining sections in Examples 1-6 and Comparative Examples 1-2. The staining results of the immunohistochemical staining sections were interpreted according to the interpretation criteria in Experimental Example 2, and the sensitivity and specificity were statistically calculated.

[0085] Table 3 Statistical calculation results of sensitivity and specificity

[0086] The statistical calculation results of sensitivity and specificity are shown in Table 3. According to the immunohistochemical staining sections prepared in Example 2, the sensitivity and specificity were statistically calculated after the staining results were interpreted. The sensitivity and specificity were higher than those in Example 1, indicating that the use of hydrazine derivative buffer and amino-polyethylene glycol-galactose liquid effectively improved the binding ability to the target antigen; the sensitivity and specificity obtained by statistical calculation according to Example 5 were higher than those in Example 2, indicating that the use of hydrazine derivative buffer and amino-polyethylene glycol-galactose liquid was combined with the use of glycerol polyoxyethylene ether cocoate to further reduce the background intensity and nonspecificity of staining, thereby improving the sensitivity and specificity; the sensitivity and specificity obtained by statistical calculation according to Comparative Examples 1 and 2 were lower than those in Example 2, indicating that the hydrazine derivative buffer and amino-polyethylene glycol-galactose liquid need to work together, and the use of any one of the hydrazine derivative buffer and amino-polyethylene glycol-galactose liquid alone had no obvious effect on improving the sensitivity and specificity of staining.

[0087] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0088] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A mixed buffer solution for preparing an endogenous enzyme inhibitor, wherein the mixed buffer solution contains a hydrazine derivative and amino-polyethylene glycol-galactose, and the mass ratio of the hydrazine derivative to the amino-polyethylene glycol-galactose in the mixed buffer solution is 1:0.2-1.

2. Use of a mixed buffer according to claim 1 in the preparation of endogenous enzyme inhibitors, characterized in that: The mixed buffer solution also contains a phosphate buffer solution, and the amount of the hydrazine derivative in the phosphate buffer solution is 0.045-0.06 g / ml.

3. Use of a mixed buffer according to claim 1 in the preparation of endogenous enzyme inhibitors, characterized in that: The hydrazine derivative is prepared from 1-hydroxy-2-naphthoic acid hydrazide and acetic anhydride, and the mass ratio of the amount of 1-hydroxy-2-naphthoic acid hydrazide used to the amount of acetic anhydride used is 1:0.45-0.

7.

4. An immunostaining reagent for detecting MHC-II expression in gastric cancer biopsy samples, comprising at least an endogenous enzyme inhibitor, wherein the endogenous enzyme inhibitor comprises a mixed buffer solution containing a hydrazine derivative and amino-polyethylene glycol-galactose, wherein the mass ratio of the hydrazine derivative used in the mixed buffer solution to the amino-polyethylene glycol-galactose used is 1:0.2-1.

5. The immunostaining reagent for detecting MHC-II expression in gastric cancer biopsy samples according to claim 4, characterized in that: The immunostaining reagent further comprises a dewaxing and hydrating treatment solution, wherein the dewaxing and hydrating treatment solution comprises xylene, 95-100% alcohol, 90-95% alcohol and 75-85% alcohol.

6. The immunostaining reagent for detecting MHC-II expression in gastric cancer biopsy samples according to claim 4, characterized in that: The immunostaining reagent also includes an antigen repair solution, which is obtained by adding citric acid and sodium citrate to water and mixing them. The mass ratio of the amount of citric acid used to the amount of sodium citrate used is 1:6-9, and the amount of citric acid added to the water is 0.0003-0.0005g / ml.

7. The immunostaining reagent for detecting MHC-II expression in gastric cancer biopsy samples according to claim 4, characterized in that: The immunostaining reagent further comprises a blocking agent, which is sheep serum.

8. The immunostaining reagent for detecting MHC-II expression in gastric cancer biopsy samples according to claim 4, characterized in that: The immunostaining reagent further comprises an enhancing solution.

9. The immunostaining reagent for detecting MHC-II expression in gastric cancer biopsy samples according to claim 4, characterized in that: The immunostaining reagent also includes a primary antibody, which is a diluted MHC Class II recombinant rabbit monoclonal antibody. The diluted MHC Class II recombinant rabbit monoclonal antibody is obtained by mixing and diluting phosphate buffer and MHC Class II recombinant rabbit monoclonal antibody at a volume ratio of 1:0.002-0.

015.

10. An immunostaining kit for detecting MHC-II expression in gastric cancer biopsy samples, comprising the immunostaining reagent according to any one of claims 4 to 9.

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