Alexa fluor 484

Double immunofluorescence was performed based on a previously reported technique for double immunohistochemistry [49 (link)] and immunofluorescence [50 (link)] with modifications. Briefly, a two-color immunofluorescence stain was performed using P-glycoprotein (green color) and AR (red color) antibodies. Tissue samples were deparaffinized, and antigen retrieval was performed using citrate buffer solution (pH 6.) in a commercial pressure chamber (Dako, Carpinteria, CA, USA) for 30 s at 125 °C, followed by 25 min at 94 °C, and cooling for 15 min. Then, unpacific binding proteins were blocked using a commercial solution for 30 min (Dako, Carpinteria, CA, USA). Anti-rabbit polyclonal AR (1:50) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alexa Fluor 484 (Invitrogen, Carlsbad, CA, USA) at a 2 μg/mL dilution in phosphate-buffered saline (PBS) for 1 h and the slides were washed using PBS solution. The second primary antibody was applied (1:700 monoclonal mouse anti-P-gp clone C494, BioLegend, San Diego, CA, USA) overnight. Then, the secondary goat anti-mouse antibody was conjugated with Alexa Fluor^® 594 (BioLegend, San Diego, CA, USA), for one hour. Slides were counterstained with 4-6-diamidino-2-phenylindole (DAPI; Sigma, Portland, OR, USA) and evaluated using a laser scanning confocal microscope (Leica Biosystems, Wetzlar, Germany).