Paraffin-embedded tissue sections were deparaffinized at 60°C for 20 min, cleared in xylene, and subjected to a graded series of alcohol. For hematoxylin and eosin (H&E) staining, slides were stained with Mayer’s hematoxylin (Sigma-Aldrich), blued in 0.1% sodium bicarbonate, and counterstained with Eosin Y solution (Sigma-Aldrich). For IHC, the slides were heated with saline sodium citrate (SSC) buffer at 95° to 100°C. After cooling, the slides were blocked with blocking solution (2% goat serum, 2% bovine serum albumin, and 0.05% Tween 20 in PBS) at room temperature and incubated with a primary antibody diluted in blocking solution at 4°C. Endogenous peroxidase activity was quenched with 0.3% H2O2. Slides were incubated with a horseradish peroxidase (HRP)–conjugated secondary antibody (GeneTech) at room temperature, developed with 3,3′-diaminobenzidine substrate (GeneTech), counterstained and blued with hematoxylin, and dehydrated with a graded series of alcohol. The positive staining density was measured using a computerized imaging system composed of a Leica CCD camera DFC420 connected to a Leica DM IRE2 microscope (Leica Microsystems Imaging Solutions Ltd.). The H-score scoring system was used, which evaluated staining intensity (0 to 3) and the percentage of positively stained cells (0 to 1), with a final score ranging from 0 to 3.

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