Improved immunohistochemical method for detecting VISTA protein in tissue slice

Abstract

The invention discloses an improved immunohistochemical method for detecting VISTA protein in a tissue slice, which further comprises a step of removing glycosylation modification of VISTA after an antigen repair step and before a peroxidase inactivation step. In the colorectal cancer tissue slice, after the glycosylation modification of the VISTA is removed, the detection rate of the VISTA in immunohistochemistry can be obviously improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection technology, and in particular relates to an improved immunohistochemical method for detecting VISTA protein in tissue sections. Background Art

[0002] VISTA (V-domain immunoglobulin suppressor of T cell activation) is a newly discovered immunosuppressive checkpoint molecule in recent years. It is similar in structure to PD-L1 and belongs to the B7 family of immune checkpoints. VISTA can be expressed in T lymphocytes (including CD4+ and CD8+ T lymphocytes), and is significantly highly expressed in myeloid-derived monocytes / macrophages, neutrophils and dendritic cells, and can also be seen in certain tumor cells. VISTA mainly inhibits T cell activation, thereby allowing tumor cells to escape the body's immune recognition and attack. Specific blocking of VISTA can significantly enhance the anti-tumor immune response. However, the traditional immunohistochemistry (IHC) method is not effective in detecting the expression of VISTA in colorectal cancer tissues. The positive detection rate of VISTA in colorectal cancer tumor cells is only 1.8-2.6%. Therefore, enhancing the sensitivity of immunohistochemistry (IHC) detection of VISTA in colorectal cancer tissues, especially improving the positive detection rate of VISTA in colorectal cancer cells, is extremely critical for accurately identifying patient groups that may benefit from VISTA monoclonal antibody treatment. Summary of the invention

[0003] To this end, in view of the technical problem of the detection sensitivity of existing immunohistochemistry (IHC) for detecting VISTA in colorectal cancer tissue, the present invention provides an improved immunohistochemistry method for detecting VISTA protein in tissue sections, characterized in that the method includes a step of removing the glycosylation modification of VISTA.

[0004] The present invention includes a step of removing the glycosylation modification of VISTA after the antigen repair step and before the peroxidase inactivation step.

[0005] Preferably, the tissue section is a tumor tissue section, preferably a colorectal cancer tissue section.

[0006] The improved immunohistochemical method for detecting VISTA protein in tissue sections of the present invention specifically comprises the following steps:

[0007] Step S1, drying and dewaxing the tissue sections;

[0008] Step S2, performing antigen retrieval on tissue sections;

[0009] Step S3, denaturing the glycoprotein and removing the glycosylation modification of VISTA;

[0010] Step S4, inactivating peroxidase;

[0011] Step S5, first blocking, then incubating with primary antibody and secondary antibody;

[0012] Step S6, color development and observation.

[0013] Preferably, the method comprises the following steps:

[0014] Step S1, drying the tissue slices at 60°C to 65°C for 85 minutes to 120 minutes, and then repeatedly soaking in an organic solvent for dewaxing;

[0015] Step S2, heating the tissue sections with citric acid antigen repair solution in a microwave oven at the thawing setting for two times for 8 to 12 minutes for antigen repair;

[0016] Step S3, incubating with glycoprotein denaturation buffer at room temperature for 2.5 to 5 hours to denature the glycoprotein; then incubating with deglycosylase diluted to 5% in buffer at 36 to 38° C. overnight to remove the glycosylation modification of VISTA;

[0017] Step S4, covering with a hydrogen peroxide solution diluted with methanol and protecting from light for 20 to 40 minutes to inactivate peroxidase;

[0018] Step S5, first blocking with 4% to 8% goat serum at room temperature for 50 to 80 minutes, then incubating with VISTA primary antibody prepared with 4% to 8% goat serum at 4°C overnight, and then incubating with mouse and rabbit universal secondary antibody at room temperature for 50 to 80 minutes after rewarming;

[0019] Step S6, color development and observation.

[0020] Preferably, the method comprises the following steps:

[0021] Step S1, drying the tumor tissue slices at 63°C for 90 minutes, and then repeatedly soaking them in an organic solvent for dewaxing;

[0022] Step S2, the tumor tissue sections are subjected to antigen repair by heating the citric acid antigen repair solution in a microwave oven twice for 10 minutes at the thawing setting;

[0023] Step S3, incubating with glycoprotein denaturation buffer at room temperature for 3 hours to denature the glycoprotein, and then incubating with deglycosylase N-glycosidase F (PNGase F) diluted to 5% with PBS at 37°C overnight to remove the glycosylation modification of VISTA;

[0024] Step S4, covering with a hydrogen peroxide solution diluted to 30% by methanol and protecting from light for 30 minutes to inactivate peroxidase;

[0025] Step S5, first blocking with 5% goat serum at room temperature for 1 hour, then incubating with VISTA primary antibody prepared with 5% goat serum at 4°C overnight, and then incubating with mouse and rabbit universal secondary antibody at room temperature for 1 hour after rewarming;

[0026] Step S6, color development and observation.

[0027] Preferably, the repeated soaking in organic solvent in step S1 specifically includes:

[0028] Soak in xylene for 5 minutes, soak in xylene twice for 5 minutes, soak in anhydrous ethanol for 5 minutes, soak in anhydrous ethanol twice for 5 minutes, soak in 95% ethanol for 3 minutes, soak in 95% ethanol twice for 3 minutes, soak in 75% ethanol for 3 minutes, and soak in 75% ethanol twice for 3 minutes.

[0029] Preferably,

[0030] In step S1, after dewaxing, washing with clean water for 4 to 10 minutes;

[0031] In step S2, after two microwave heatings, the samples are cooled to room temperature respectively; after antigen repair, the samples are washed with buffer for 3 to 5 times, each time for 4 to 10 minutes;

[0032] In step S3, the glycoprotein denaturation buffer is a 0.5% SDS (Sodium dodecyl sulfate) solution containing 40 mM DTT (Dithiothreitol), and the glycoprotein is washed with the buffer for 3 to 5 times, each time for 4 to 10 minutes after denaturation; after removing the glycosylation modification of VISTA, the buffer is washed for 3 to 5 times, each time for 4 to 10 minutes;

[0033] In step S4, after inactivation in dark, washing is performed with a buffer solution for 3 to 5 times, each time for 4 to 10 minutes.

[0034] Wherein, the buffer solution is PBS buffer solution.

[0035] The color observation includes: DAB color development, hematoxylin counterstaining, transparent sealing and microscopic observation and photography.

[0036] The positive and progressive effect of the present invention is that after removing the glycosylation modification of VISTA in the colorectal cancer tissue sections of the present invention, the detection rate of VISTA in immunohistochemistry can be significantly improved. Through deglycosylation treatment, the expression of VISTA in colorectal cancer tissue can be more effectively detected, which is extremely critical for accurately identifying the patient population that may benefit from VISTA monoclonal antibody treatment.

[0037] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1A Analyze the expression of VISTA in various cell subpopulations for single-cell data;

[0039] Figure 1B Analyze the expression of VISTA in pan-cancer cell lines for the CCLE database;

[0040] Figure 1C Western blots were used to detect the protein expression of VISTA in a panel of colorectal cancer cell lines;

[0041] Figure 2A Western blots were used to detect changes in protein expression of VISTA in colorectal cancer cell lines after PNGase F treatment;

[0042] Figure 2B Western blots were used to detect changes in VISTA protein expression in lung cancer and prostate cancer cell lines after PNGase F treatment;

[0043] Figure 2C After PNGase F treatment, the expression changes of VSITA in colorectal cancer cells were detected by cytofluorescence (IF);

[0044] Figure 2D After treatment with the glycosylase inhibitor TM, the protein expression changes of VISTA in colorectal cancer cell lines were detected;

[0045] Figure 2E After treatment with glycosylase inhibitors NGI-1 and TM, the expression changes of VSITA in colorectal cancer cells were detected by cytofluorescence (IF);

[0046] Figure 3A and Figure 3B Representative images (scale bar = 20 μm) and H-score change graphs of VISTA staining intensity changes in tumor cells and stromal cells in colorectal cancer tissues treated with PNGase F deglycosylation compared with those without treatment, respectively;

[0047] Figure 3C and Figure 3D These are the trends of VISTA staining changes in tumor cells and stromal cells in colorectal cancer tissues treated with PNGase F deglycosylation compared with those without treatment;

[0048] Figure 3EThis is the relationship between the difference in VISTA expression and the OS and DFS of patients after PNGase F deglycosylation treatment. DETAILED DESCRIPTION

[0049] In response to the phenomenon that "the positive detection rate of VISTA in colorectal cancer tumor cells is only 1.8-2.6%" (refer to Zong L, Yu S, Mo S, Zhou Y, Xiang Y, Lu Z, et al. High VISTA Expression Correlates With a Favorable Prognosis in Patients With Colorectal Cancer. Journal of immunotherapy (Hagerstown, Md: 1997), 2021; 1: 22-28.), the applicant first detected the expression levels of VISTA mRNA and protein in colorectal cancer tumor cells to determine whether the low IHC detection rate of VISTA in colorectal cancer tumor cells is due to its own low expression or because other reasons affect the IHC detection efficiency.

[0050] By analyzing the single-cell public database, the applicant found that VISTA was transcriptionally activated in 13.1% of colorectal cancer tumor cells ( Figure 1A ), and through pan-cancer tumor cell transcriptome analysis, it was found that VISTA is expressed in various tumor cells including colorectal cancer ( Figure 1B ). Subsequently, at the protein level, the applicant found that all colorectal cancer cell lines highly expressed VISTA, but from the Western Blots images, it can be clearly found that the VISTA protein is dragged, suggesting that VISTA has post-translational modification ( Figure 1C ). So could it be that post-translational modification of the protein affects the detection rate of VISTA in IHC? The applicant made various attempts and surprisingly found that removing the glycosylation level of VISTA can effectively increase the IHC positive rate of VISTA in colorectal cancer tumor cells. The specific embodiment process is listed as follows:

[0051] Example 1 IHC detection of VISTA in colorectal cancer tissue sections

[0052] Slice baking: Insert the colorectal cancer tissue slices into the slice rack and bake them in a 63°C oven for at least 90 minutes.

[0053] Dewaxing: Soak the baked tissue sections in the following order: xylene No. 1 (5 minutes) → xylene No. 2 (5 minutes) → anhydrous ethanol No. 1 (5 minutes) → anhydrous ethanol No. 2 (5 minutes) → 95% ethanol No. 1 (3 minutes) → 95% ethanol No. 2 (3 minutes) → 75% ethanol No. 1 (3 minutes) → 75% ethanol No. 2 (3 minutes). After the end, wash with plenty of water for 5 minutes.

[0054] Antigen repair: Place tissue sections in a histochemical box, fill with citric acid antigen repair solution, heat in a microwave oven for 10 minutes, open the microwave door, let it cool naturally for 5 minutes, heat in the thawing mode for another 10 minutes, then take out the histochemical box and place it in water to cool down. When the temperature drops to room temperature, wash with PBS 3 times, 5 minutes each time.

[0055] Glycoprotein denaturation: The tissue sections were incubated with 1× glycoprotein denaturation buffer (0.5% SDS, 40 mM DTT) at room temperature for 3 hours, and then washed 4 times with PBS for 5 minutes each time.

[0056] Deglycosylation: Tissue sections were incubated with PNGase F (NEB, #P0704L) diluted to 5% in PBS at 37°C overnight, and then washed three times with PBS for 5 minutes each time.

[0057] Inactivation: First, circle the tissue area with an immunohistochemistry brush, then cover the tissue area with 30% hydrogen peroxide diluted to 3% with methanol, inactivate peroxidase for 30 minutes in the dark, and then wash with PBS 3 times, 5 minutes each time.

[0058] Blocking: Cover the tissue with 5% goat serum for blocking at room temperature for 1 hour.

[0059] Incubate with primary antibody: shake off the goat serum used for blocking on the slices, prepare VISTA primary antibody with 5% goat serum (volume ratio of 1:100, antibody information: CST, #64953), place in a humidified box, and incubate overnight at 4°C.

[0060] Rewarming: Take the wet box containing the slices out of the 4°C refrigerator, open the lid, and let it stand at room temperature for about 30 minutes to allow it to rewarm naturally.

[0061] Incubation with secondary antibody: Wash the rewarmed sections with PBS three times, 10 minutes each time. Then drop the mouse and rabbit universal secondary antibody (Gene Technology (Shanghai) Co., Ltd.) on the tissue and incubate at room temperature for 1 hour.

[0062] DAB color development: After removing the secondary antibody, the sections were washed with PBS for 3 times, 10 minutes each time. DAB color developer (Gene Technology (Shanghai) Co., Ltd.) was prepared at a volume ratio of 1:25 and color was developed under a microscope.

[0063] Hematoxylin counterstaining: Stain the tissue area in the hematoxylin-covered section for 1 minute, then rinse with plenty of water for 20 minutes.

[0064] Transparent sealing: Soak the tissue sections in the following order: 75% ethanol No. 1 (5 minutes) → 75% ethanol No. 2 (5 minutes) → 95% ethanol No. 1 (5 minutes) → 95% ethanol No. 2 (5 minutes) → anhydrous ethanol No. 1 (3 minutes) → anhydrous ethanol No. 2 (5 minutes) → xylene No. 1 (10 minutes) → xylene No. 2 (10 minutes). Finally, seal the sections with neutral resin.

[0065] Photography: After the slides have been air-dried for 24 hours, they can be used for observation and photography under a microscope.

[0066] Example 2 Western Blots Detection of VISTA in Colorectal Cell Lines

[0067] Prepare protein lysis buffer: add PMSF (20 mg / mL), sodium fluoride (100 mmoL), sodium vanadate (200 mmoL), and protease inhibitor (PI) to 1% NP40 at a volume ratio of 1:100, and shake gently to make it uniform.

[0068] Extract cell protein: Collect the culture dish where the adherent cells are to be extracted, remove the supernatant, wash twice with PBS to remove the residual culture medium, add about 200-500 μL lysis solution to the culture dish, scrape the cells from the culture dish,

[0069] Transfer to a 1.5 mL EP tube and perform ultrasonic lysis.

[0070] Glycoprotein denaturation: Take 18 μL of cell lysate, add 2 μL of 10× glycoprotein denaturation buffer (5% SDS, 400 mM DTT), mix by pipetting, incubate in a 100°C metal bath for 10 minutes, and then quickly cool on ice.

[0071] Deglycosylation: Take 10 μL of glycoprotein denatured product, add 2 μL of 500 mM sodium phosphate buffer (pH 7.5), 2 μL of 10% NP40 and 6 μL of double distilled water to make a 20 μL reaction system, then add 1 μL of PNGase F and incubate at 37°C overnight.

[0072] Protein concentration determination: According to the Bradford kit instructions, add the protein supernatant obtained by centrifugation and G250 (detergent compatible) reagent at a volume ratio of 10:300, and gently pipette evenly. Add 300 μL of the mixture to each well of the 96-well plate, then measure the absorbance (OD value) at 595 nm with an ELISA reader, calculate the protein concentration according to the standard curve, and mark the tube.

[0073] Gel preparation: Use the rapid gel preparation kit of Shanghai Yazyme Biopharmaceutical Technology Co., Ltd. to prepare separation gel of corresponding concentration.

[0074] Electrophoresis: Fix the gel in the electrophoresis tank, add enough electrophoresis solution, and then add the protein sample to the corresponding electrophoresis tank. Use 80V voltage in the concentrated gel, and when the protein and marker are separated and enter the lower separation gel, use 120V voltage for electrophoresis.

[0075] Transfer: Place the transfer sponge, transfer filter paper, glue, NC membrane, transfer filter paper and transfer sponge in order, clamp them with transfer clips, insert them into the transfer tank, add ice box, and fill with transfer solution. Transfer at 300mA for 1 hour under constant current conditions.

[0076] Blocking: Soak the transferred NC membrane in 5% skim milk and block it at room temperature for 1 hour.

[0077] Incubate with primary antibody: Cut the target band area from the NC membrane, soak it in 5% skim milk containing VISTA primary antibody (CST, #64953) (primary antibody concentration 1:1000), place it on a shaker, and incubate it at 4°C overnight or at room temperature for 2 hours.

[0078] Incubate with secondary antibody: Recover the 5% skim milk diluted with primary antibody, wash the NC membrane with TBST for 3 times, 5 minutes each time, then prepare the secondary antibody corresponding to the primary antibody with TBST, place on a shaker, and incubate at room temperature for 1 hour.

[0079] Washing: After the secondary antibody incubation, the NC membrane was washed three times with TBST, 5 minutes each time.

[0080] Development: Prepare Millipore ECL developer in a 1:1 volume ratio and place in a luminometer for development.

[0081] Example 3IF detection of VISTA in colorectal cancer cell lines

[0082] Slide preparation: Colorectal cancer cells were inoculated into a 24-well plate with slides placed in advance. After 2 days, the cells grew on the slides and the culture medium in the wells was aspirated and the plates were washed 3 times with PBS.

[0083] Fixation: Add 1 mL of 4% paraformaldehyde for fixation at room temperature for 15 minutes, and then wash three times with PBS.

[0084] Glycoprotein denaturation: Add 1× glycoprotein denaturation buffer (0.5% SDS, 40 mM DTT) to the wells, incubate at 100°C for 10 minutes, then quickly cool on ice, and then wash three times with PBS.

[0085] Deglycosylation treatment: Incubate the slides with 5% PNGase F diluted in PBS at 37°C overnight, then wash three times with PBS.

[0086] Permeabilization: Add 1 mL of 0.2% TritonX-100 and perform permeabilization at room temperature for 10 minutes, then wash three times with PBS.

[0087] Blocking: Add 1 mL of 5% goat serum for blocking at room temperature for 1 hour, and then wash three times with PBS.

[0088] Antibody incubation: VISTA primary antibody (CST, #64953) was added to 5% goat serum at a volume ratio of 100:1, incubated at room temperature for 1 hour, washed 3 times with PBS, and then added with the corresponding secondary antibody and incubated at room temperature for 1 hour, and then washed 3 times with PBS.

[0089] Sealing observation: Use DAPI to stain for 15 minutes in the dark, wash with PBS three times, and then add anti-fluorescence quencher to seal the slide. Observe the expression of VISTA in cells under a fluorescence microscope.

[0090] Comparative Example 1

[0091] Except for the absence of the glycoprotein denaturation step and the deglycosylation step, the other steps are the same as those in Example 1.

[0092] As can be seen from Example 2, after the colorectal cancer cell line was treated with the deglycosylation enzyme PNGase F, the VISTA protein was clearly detected to be converted from a 40-70 kD band to a bright band of about 36 kD, which improved the detection efficiency of Western blots ( Figure 2A This phenomenon also exists in non-colorectal cancer cells, such as lung cancer and prostate cancer ( Figure 2B ). At the same time, Example 3 can clearly detect that after the colorectal cancer cell line is treated with PNGase F, the fluorescence intensity of VISTA is significantly enhanced ( Figure 2C ). In addition, treatment with glycosylase inhibitors NGI-1 and TM (Tunicamycin) can also significantly enhance the detection efficiency of VISTA in colorectal cancer cells. ( Figure 2D , Figure 2E ).

[0093] Combined with the above experimental results, the applicant found that removing the glycosylation level of VISTA can effectively improve the detection intensity of VISTA in colorectal cancer tumor cells in Western blots and IF. In the subsequent IHC detection process (according to the detection method of Example 1 and Comparative Example 1), the applicant used the deglycosylation enzyme PNGase F to treat the tissue sections, and it was found that the VISTA positive rate of colorectal cancer tumor cells was significantly improved (from 2.63% to 20.5%). At the same time, the VISTA of stromal cells was also improved to a certain extent (from 35.8% to 59.5%) (see Figures 3A to 3E ).

[0094] Effect Example

[0095] 190 cases of colorectal cancer tissue sections were dried, dewaxed, and antigen repaired according to the method steps of Example 1, and then the sections were denatured for glycoproteins using 1× glycoprotein denaturation buffer (0.5% SDS, 40mM DTT), and then deglycosylated using 5% PNGase F, followed by inactivation, blocking, and primary antibody application. Corresponding colorectal cancer tissue sections were dried, dewaxed, antigen repaired, inactivated, blocked, and primary antibody applied according to the method steps of Comparative Example 1.

[0096] According to the immunohistochemistry results, the applicant subsequently performed a tissue score (H-score), which is calculated by multiplying the staining intensity (0, negative; 1, weak; 2, moderate; 3, strong) and the percentage of positive staining (the total score range is 0-300). After deglycosylation treatment, the VISTA positive rate was significantly improved, especially the VISTA positive rate of colorectal cancer tumor cells increased from 2.63% to 20.5% ( Figures 3A to 3D ). Moreover, after deglycosylation treatment, the expression of VISTA in colorectal cancer cells is an important marker for poor prognosis of colorectal cancer ( Figure 3E ), which is crucial for the subsequent screening of people who will benefit from VISTA monoclonal antibodies.

Claims

1. An improved immunohistochemical method for detecting VISTA protein in tissue sections, characterized in that The method includes the step of removing the glycosylation modification of VISTA.

2. The improved immunohistochemical method for detecting VISTA protein in tissue sections as claimed in claim 1, characterized in that After the antigen retrieval step and before the peroxidase inactivation step, a step to remove the glycosylation modification of VISTA is included.

3. The improved immunohistochemical method for detecting VISTA protein in tissue sections as described in claim 1 or 2, characterized in that The tissue slice is a tumor tissue slice, preferably a colorectal cancer tissue slice.

4. The improved immunohistochemical method for detecting VISTA protein in tissue sections as claimed in claim 2, characterized in that The method comprises the following steps: Step S1, drying and dewaxing the tissue sections; Step S2, performing antigen retrieval on tissue sections; Step S3, denaturing the glycoprotein and removing the glycosylation modification of VISTA; Step S4, inactivating peroxidase; Step S5, first blocking, then incubating with primary antibody and secondary antibody; Step S6, color development and observation.

5. The improved immunohistochemical method for detecting VISTA protein in tissue sections as claimed in claim 4, characterized in that The method comprises the following steps: Step S1, drying the tissue slices at 60°C to 65°C for 85 minutes to 120 minutes, and then repeatedly soaking in an organic solvent for dewaxing; Step S2, heating the tissue sections with citric acid antigen repair solution in a microwave oven at the thawing setting for two times for 8 to 12 minutes for antigen repair; Step S3, incubating with glycoprotein denaturation buffer at room temperature for 2.5 to 5 hours to denature the glycoprotein; then incubating with deglycosylase diluted to 5% in buffer at 36 to 38° C. overnight to remove the glycosylation modification of VISTA; Step S4, covering with a hydrogen peroxide solution diluted with methanol and protecting from light for 20 to 40 minutes to inactivate peroxidase; Step S5, first blocking with 4% to 8% goat serum at room temperature for 50 to 80 minutes, then incubating with VISTA primary antibody prepared with 4% to 8% goat serum at 4°C overnight, and then incubating with mouse and rabbit universal secondary antibody at room temperature for 50 to 80 minutes after rewarming; Step S6, color development and observation.

6. The improved immunohistochemical method for detecting VISTA protein in tissue sections as claimed in claim 5, characterized in that The method comprises the following steps: Step S1, drying the tumor tissue slices at 63°C for 90 minutes, and then repeatedly soaking them in an organic solvent for dewaxing; Step S2, the tumor tissue sections are subjected to antigen repair by heating the citric acid antigen repair solution in a microwave oven twice for 10 minutes at the thawing setting; Step S3, incubating with glycoprotein denaturation buffer at room temperature for 3 hours to denature the glycoprotein, and then incubating with deglycosylase N-glycosidase F diluted to 5% with PBS at 37°C overnight to remove the glycosylation modification of VISTA; Step S4, covering with a hydrogen peroxide solution diluted to 30% by methanol and protecting from light for 30 minutes to inactivate peroxidase; Step S5, first blocking with 5% goat serum at room temperature for 1 hour, then incubating with VISTA primary antibody prepared with 5% goat serum at 4°C overnight, and then incubating with mouse and rabbit universal secondary antibody at room temperature for 1 hour after rewarming; Step S6, color development and observation.

7. The improved immunohistochemical method for detecting VISTA protein in tissue sections as claimed in claim 6, characterized in that The repeated soaking with organic solvent in step S1 specifically includes: Soak in xylene for 5 minutes, soak in xylene twice for 5 minutes, soak in anhydrous ethanol for 5 minutes, soak in anhydrous ethanol twice for 5 minutes, soak in 95% ethanol for 3 minutes, soak in 95% ethanol twice for 3 minutes, soak in 75% ethanol for 3 minutes, and soak in 75% ethanol twice for 3 minutes.

8. The improved immunohistochemical method for detecting VISTA protein in tissue sections as claimed in claim 6, characterized in that In step S1, after dewaxing, washing with clean water for 4 to 10 minutes; In step S2, after two microwave heatings, the samples are cooled to room temperature respectively; after antigen repair, the samples are washed with buffer for 3 to 5 times, each time for 4 to 10 minutes; In step S3, the glycoprotein denaturation buffer is a 0.5% sodium dodecyl sulfate solution containing 40 mM dithiothreitol, and the glycoprotein is washed with the buffer for 3 to 5 times, each time for 4 to 10 minutes after denaturation; after removing the glycosylation modification of VISTA, the buffer is washed for 3 to 5 times, each time for 4 to 10 minutes; In step S4, after inactivation in dark, washing is performed with a buffer solution for 3 to 5 times, each time for 4 to 10 minutes.

9. The improved immunohistochemical method for detecting VISTA protein in tissue sections as claimed in claim 8, characterized in that The buffer is PBS buffer.

10. The improved immunohistochemical method for detecting VISTA protein in tissue sections as claimed in claim 5, characterized in that The color observation includes: DAB color development, hematoxylin counterstaining, transparent sealing and microscopic observation and photography.

Images