The immunohistochemical procedure is carried out in the following steps: First, tissue samples are collected from both the experimental and control groups. It is important to keep the tissues as fresh as possible. After collection, the tissues are rinsed with phosphate-buffered saline (PBS) and cut into small blocks measuring less than 0.5 cm × 0.5 cm × 0.1 cm. Next, the tissues are fixed in 4% paraformaldehyde for sufficient time to preserve cellular structures. Following fixation, the tissues are dehydrated through a graded ethanol series: 70% ethanol for 1 hour, 80% ethanol for 1 hour, 95% ethanol for 1.5 hours, repeated twice, followed by 100% ethanol for 1 hour, also repeated twice. After dehydration, the tissues are cleared using xylene for 20 minutes, twice. Then, they are infiltrated with paraffin at 65°C for 45 minutes, repeated twice. Finally, the tissues are embedded in a copper mold to form solid blocks. Once embedded, 5-µm thick sections are cut and mounted onto poly-L-lysine-coated glass slides. The slides are then baked at 65°C for 4.5 hours to ensure proper adhesion of the tissue sections. Before proceeding with staining, the sections are dewaxed and rehydrated. This involves immersing the slides in xylene for 1.5 hours (this step can be adjusted if needed), followed by a series of ethanol washes: 100%, 95%, and 80% ethanol, each for a few minutes. The slides are then rinsed with tap water. Next, the slides are incubated with 3% methanol-hydrogen peroxide solution at room temperature for 30 minutes to block endogenous peroxidase activity. After washing with tap water, antigen retrieval is performed using a heat-induced method. This involves placing an iron pot in a microwave oven, adding distilled water to the bottom of the pot, and inserting a glass beaker containing a citrate buffer solution. The water is heated until boiling, and the timer is started for 3–5 minutes. Afterward, the heat is turned off, and the slides are cooled in cold water until they reach room temperature. Finally, the slides are washed with PBS before proceeding to the next staining steps. This detailed process ensures that the tissue samples are properly prepared for immunohistochemical analysis, allowing for accurate detection of target proteins.

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