Vectastain system

Brains were fixed with 4% paraformaldehyde, dehydrated in 30% sucrose and stained using the Vectastain system (Vector Labs, Burlingame, CA, USA), as described previously.^{57 (link)} Immunostaining with mouse anti-NR2B (1:2000; Abcam, Cambridge, MA, USA), followed by a secondary goat anti-mouse antibody and avidin–biotin complex, was visualized with diaminobenzidine. Sections were observed with a light microscope (Leica, Buffalo Grove, IL, USA) to verify the expression of NR2B in RSC.

FDA-grade human tissue microarray is from US Biomatrix (Rockville, MD). Patient radical prostatectomy specimens were from New York University Medical Center, NY. Tissues slides were rehydrated in xylene and a series of graded ethanol. Antigen retrieval was performed with 0.01 M citrate buffer at pH 6.0 for 20 minutes at 95°C. Slides were allowed to cool for another 30 minutes, followed by sequential rinsing in phosphate-buffered saline with 0.01% Triton X (PBS-T). Endogenous peroxidase activity was quenched by incubation in PBS-T containing 3% hydrogen peroxide. Each incubation step was carried out at room temperature and was followed by three sequential washes in PBS-T. Sections were incubated in 5% goat serum albumin in RT for one hour before rabbit polyclonal anti-DNAH8 antibody (Sigma-Aldrich) for overnight at 4°C. The next day, slides were washed with PBS-T three times and were incubated with biotinylated secondary antibody for 1 hour, peroxidase-labeled streptavidin (Vectastain system, Vector Laboratories) for 1 hour, and diaminobenzidine substrate for peroxidase-based immunohistochemistry (Cardassian DAB Chromogen, Biocare Medical). Slides were counter-stained with hematoxylin (Vector Laboratories) and dehydrated before mounted.