2.5 Detection of Mutant p53 Expression in Tumor Tissue Paraffin Sections Using Immunohistochemistry

2.5 Detection of Mutant p53 Expression in Tumor Tissue Paraffin Sections Using Immunohistochemistry

2.5.1 Preparation of Paraffin Sections

Sectioning: Tissue embedded in paraffin was cut into 5μm thick paraffin sections using a pathology sectioning machine.

De-waxing and hydration: Before de-waxing, the sections were baked in a 45℃ constant temperature oven for 120 minutes. The sections were then immersed in xylene for 10 minutes, followed by another 10-minute immersion in fresh xylene, and subsequently soaked in anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, and 70% ethanol for 5 minutes.

2.5.2 Immunohistochemical Staining (SP Method)

• Wash the sections with PBS 2–3 times, 5 minutes each
• Add 3% H₂O₂ to eliminate endogenous peroxidase activity, let stand at room temperature for 10 minutes
• Wash the sections with PBS 2–3 times, 5 minutes each
• Antigen retrieval: Heat a 0.01M sodium citrate buffer solution (pH 6.0) in a water bath to about 92–95℃, place the sections in the solution for 15 minutes, then allow to cool naturally.
• Apply normal goat serum blocking solution, let sit at room temperature for 15 minutes, then shake off excess liquid
• Add primary antibody (rabbit anti-human P53 monoclonal antibody), incubate at 37℃ for 2.5 hours
• PBS wash, 3 min × 3 times
• Add biotinylated secondary antibody working solution, incubate at 37°C for 15 min
• PBS wash, 3 min × 3 times
• Add horseradish peroxidase-conjugated streptavidin working solution, incubate at 37°C for 15 min
• PBS wash, 3 min × 3 times
• DAB staining: 1 ml distilled water + one drop each of ABC three liquids (prepare fresh before use), stain for 5–10 min, observe the staining degree under a microscope
• PBS wash for 10 min
• Hematoxylin counterstain
• Stop the reaction by rinsing with tap water
• Sequential dehydration, xylene clearing, and mounting with neutral gum

2.5.3 p53 positive judgment criteria

The positive reaction product of mutant p53 protein is mainly located in the nucleus; when the nucleus is stained brown or brownish-yellow, it is considered a positive reaction for mutant p53, otherwise it is negative. Randomly observe 10 high-power fields and calculate the number of positive cells among 100 tumor cells in each field, then take the average as the number of p53-positive cells.