2.6 Detection of VEGF and p53 Expression in Tumor Tissue Paraffin Sections Using Immunohistochemistry

2.6 Detection of VEGF and p53 Expression in Tumor Tissue Paraffin Sections Using Immunohistochemistry

After the experiment, take out the tumor tissue, fix it with 10% formaldehyde, dehydrate it, and embed it in paraffin.

2.6.1 Preparation of Paraffin Sections

Sectioning: Use a pathological microtome to cut the paraffin-embedded tissue into 5 μm thick paraffin sections.

De-waxing and hydration: Before de-waxing, bake the sections in a 45°C constant-temperature oven for 60 minutes. Soak the sections in xylene for 20 minutes, replace the xylene and soak again for another 20 minutes, then sequentially soak in anhydrous ethanol for 10 minutes, 95% ethanol for 10 minutes, 90% ethanol for 10 minutes, and 85% ethanol for 10 minutes.

2.6.2 Immunohistochemical Staining (SABC Method)

• Wash with PBS 2–3 times, 5 minutes each
• Add 3% H₂O₂ and let it sit at room temperature for 10 minutes to eliminate endogenous peroxidase activity
• Wash with PBS 2–3 times, 5 minutes each
• Antigen retrieval: Heat a 0.01 M sodium citrate buffer solution (pH 6.0) in a water bath to about 92°C–95°C, place the sections in it and boil for 15 minutes. Allow to cool naturally for 20–30 minutes
• Rinse with PBS (pH 7.2–7.6), 3 minutes × 3 times
• Apply normal goat serum blocking solution, let it sit at room temperature for 15 minutes, shake off excess liquid, do not wash
• Add primary antibodies (rabbit anti-mouse VEGF monoclonal antibody, rabbit anti-mouse p53 monoclonal antibody) separately, incubate at 37°C for about 2 hours
• Rinse with PBS (pH 7.2–7.6), 3 minutes × 3 times
• Add biotinylated secondary antibody working solution separately, incubate at 37°C for 15 minutes
• Rinse with PBS, 3 minutes × 3 times
• Add horseradish peroxidase-labeled streptavidin working solution separately, incubate at 37°C for 15 minutes
• Rinse with PBS, 3 minutes × 3 times
• DAB staining: Use the DAB chromogenic kit. Take 1 ml of distilled water, add one drop each of reagents A, B, and C from the kit, mix well, and apply to the sections. Let it develop at room temperature, control the reaction time under the microscope, generally between 5 and 30 minutes. Wash with distilled water
• Lightly counterstain with hematoxylin
• Stop the reaction by rinsing with tap water
• Dehydrate step by step, make transparent with xylene, seal with neutral gum
• Observe under the microscope

2.6.3 Immunohistochemical Index Detection

P53 positive reaction products are mainly located in the nucleus; when the nucleus is stained brown or yellow, it indicates positive expression of P53. VEGF positive reaction products are mainly located in the cytoplasm; when the cytoplasm is stained brown or yellow, it indicates positive expression of VEGF. Under a 400x optical microscope, randomly select 5 sections from each group, randomly sample 3 fields of view from each section, totaling 15 fields of view per group, capture images, and use the BI-2000 medical image analysis software to perform semi-quantitative detection of the average optical density (MOD) of VEGF and P53 stained positive substances, representing the content of VEGF and P53 respectively.