D3308

Experimental protocols were conducted according to a previous description.⁴⁵ (link) In brief, His-CD63 recombinant protein (11271-H08H; Sino Biological, China) was diluted to 1 μg/mL with binding buffer, and 200 μL of the solution was added to a 96-well microtiter plate (NUNC, Rochester, NY, USA) and then incubated overnight at 4°C. Then the wells were washed four times with washing buffer and blocked with 1% BSA in PBS for 2 h at 37°C and then washed three times with washing buffer. Afterward, biotin-labeled aptamer LL4A or Library (Sangon Biotech, China) was denatured for 10 min at 95°C and cooled for 10 min on ice, then diluted in binding buffer at concentrations indicated in the figures. Next, 200 μL of the solution was added to each well, and the plate was incubated at 37°C for 1 h and then washed four times with washing buffer to remove unbound ssDNA. Afterward, 200 μL of a 1:2,000 dilution of streptavidin labeled with horseradish peroxidase (D3308; Beyotime, China) was added to each well, and then the plate was incubated at room temperature for 2 h. At last, the plate was washed four times and developed by EL-ABTS Chromogenic Reagent kit (C510031; Sangon Biotech, China), and then 405-nm single-wavelength absorbance was detected by using a microplate reader.

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)).