2.5 Detection of P27 Protein Expression in Tumor Tissue Paraffin Sections Using Immunohistochemistry

2.5 Detection of P27 Protein Expression in Tumor Tissue Paraffin Sections Using Immunohistochemistry

2.5.1 Preparation of Paraffin Sections

Fixation: Tumor tissue was fixed with 10% formaldehyde.

Dehydration: Dehydrated and embedded in paraffin.

Sectioning: The paraffin-embedded tissue was cut into 5μm thick paraffin sections using a pathology slicer.

Deparaffinization and Hydration: Before deparaffinization, the sections were baked in a 45℃ constant temperature oven for 60 minutes. The sections were then immersed in xylene for 20 minutes, followed by another 20-minute immersion in fresh xylene, and subsequently soaked 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)

Add 3% H₂O₂ and let it sit at room temperature for 5–10 minutes to eliminate endogenous peroxidase activity. Wash three times with distilled water.

Heat-induced antigen retrieval: Immerse the sections in a container filled with 0.01M citrate buffer solution (pH 6.0), place the container in a larger vessel filled with tap water, heat to boiling on an electric stove, start timing when the temperature of the small container reaches 92–98℃, maintain for 15–20 minutes, then remove from the stove and allow to cool at room temperature for 20–30 minutes. Wash three times with PBS (pH 7.2–7.6) for 3 minutes each.

Add 5% BSA blocking solution and let it sit at room temperature for 20 minutes. Shake off excess liquid without washing.

Add appropriately diluted primary antibody (rabbit anti-mouse P27 monoclonal antibody), incubate at 37℃ for about 1 hour. Wash three times with PBS (pH 7.2–7.6) for 3 minutes each.

Add biotinylated secondary antibody working solution, incubate at 37℃ for 20 minutes. Wash three times with PBS for 3 minutes each.

Add SABC reagent, incubate at 37℃ for 20 minutes. Wash five times with PBS for 5 minutes each.

DAB staining: Use the DAB chromogenic kit (AR1022). Take 1ml distilled water, add one drop each of reagents A, B, and C from the kit, mix well, and apply to the sections. Allow to 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.

Perform stepwise dehydration, xylene clearing, and mounting with neutral gum.

Observe under the microscope.

2.5.3 Criteria for Positive P27 Protein Reaction

Positive P27 protein reactions are mainly located in the nucleus/cytoplasm. Brownish-yellow or brownish-brown granules in the nucleus/cytoplasm indicate a positive P27 reaction. Randomly observe 10 high-magnification fields of view, calculate the number of positive cells among 100 tumor cells in each field, and take the average as the number of P27-positive cells.