2.5 Detection of VEGF Expression in Tumor Tissue Paraffin Sections Using Immunohistochemistry

2.5 Detection of VEGF Expression in Tumor Tissue Paraffin Sections Using Immunohistochemistry

Twenty-four hours after the last dose, weigh the mice, then euthanize them by decapitation, remove the tumor tissue, fix it with 10% formaldehyde, dehydrate it, and embed it in paraffin.

2.5.1 Preparation of Paraffin Sections

Sectioning: Cut the paraffin-embedded tissue into 5μm thick paraffin sections using a pathology sectioning machine.

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

2.5.2 Immunohistochemical Staining (SP Method)

1. Wash with PBS 2–3 times, 5 minutes each time.
2. Add 3% H₂O₂ to eliminate endogenous peroxidase activity, let stand at room temperature for 10–15 minutes. Wash with PBS 2–3 times, 5 minutes each time.
3. Antigen retrieval: Heat a 0.01M sodium citrate buffer solution (pH 6.0) to about 92–95℃ in a water bath, place the sections inside and heat for 15 minutes, then allow to cool naturally.
4. Add normal goat serum blocking solution, let stand at room temperature for 15 minutes, then shake off excess liquid.
5. Add primary antibody (rabbit anti-mouse VEGF monoclonal antibody), incubate at 37℃ for about 2 hours. Wash with PBS 3 times, 3 minutes each time.
6. Add biotinylated secondary antibody working solution, incubate at 37℃ for 15 minutes. Wash with PBS 3 times, 3 minutes each time.
7. Add horseradish peroxidase-labeled streptavidin working solution, incubate at 37℃ for 15 minutes. Wash with PBS 3 times, 3 minutes each time.
8. DAB staining: Use the DAB chromogenic kit. Take 1ml of distilled water, add one drop each of reagents A, B, and C from the kit, mix well, then apply to the sections. Let develop at room temperature, control the reaction time under the microscope, generally between 5 and 30 minutes. Rinse with distilled water.
9. Lightly counterstain with hematoxylin.
10. Stop the reaction by rinsing with tap water.
11. Dehydrate step by step, make transparent with xylene, seal with neutral gum.
12. Observe under the microscope.

2.5.3 Criteria for Determining Positive VEGF in Tumor Tissue

The main product of positive VEGF reaction is located in the cytoplasm; when the cytoplasm stains brown or brownish-yellow, it is considered a positive VEGF reaction, otherwise negative. Randomly observe 10 high-magnification fields, calculate the average number of positive cells among 100 tumor cells in each field, and take this average as the number of VEGF-positive cells [20].