Immunohistochemical Analysis of T-cell Subsets

The prostate tissue was fixed with 4% formaldehyde for 15 min at room temperature, then washed 3 times with PBS before permeabilization with 0.2% Triton X-100 (PBS) for 10 min, also at room temperature. The paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol solutions and then incubated for 20 min in 3% H₂O₂ to quench the endogenous peroxidase activity. Next, the sections were heated in target retrieval solution (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) for 15 min in a microwave oven (Oriental Rotor, Tokyo, Japan) to retrieve the antigen. Subsequently, the sections were blocked with 2% BSA at 4°C for 15 min (D3308; Beyotime Institute of Biotechnology) and incubated with CD4+ (AF1792) and CD8+ (AF1417) primary antibodies (1:100; Beyotime Institute of Biotechnology) overnight at 4°C, and stained with a fluorescein-conjugated secondary antibody anti-CD4+-FITC antibody (F1773) and anti-CD8+-FITC antibody (F0772; 1:100; Sigma-Aldrich; Merck KGaA) for 2 h at room temperature. Finally, images were captured with Leica SP5 AOBS confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany), and the number of positive cells and field area ratio were calculated with Image J software version 1.48 (National Institutes of Health, Bethesda, MD, USA) (14 (link)).