Biotinylated horse anti mouse antibody

All chemicals were purchased from Sigma (Zwijndrecht, the Netherlands) unless indicated otherwise. The polyclonal antibody against 3β hydroxysteroid dehydrogenase (HSD3B) was a kind gift from the late Dr. Payne (Stanford, CA, USA). Biotinylated horse-anti-mouse antibody, alkaline phosphatase goat-anti-rabbit antibody, and Vectastain ABC-kit Elite were purchased from Vector Laboratories (Burlingame, CA, USA). Mouse-anti-bromodeoxyuridine (BrdU) and sheep-anti-BrdU antibodies were obtained from Beckton and Dickinson (lot nr 10100, Mountain View, CA, USA) and Abcam (ab1893, Cambridge, England), respectively. Polyclonal anti-SOX9 rabbit antibody was purchased from Millipore (lot nr 2065999, Temecula, CA, USA), whereas the secondary fluorescent antibodies anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 594 were obtained from Abcam; DAPI was obtained from Sigma (D9564, St. Louis, MO, USA). Acetylated BSA (BSA-c) was purchased from Aurion (Wageningen, the Netherlands). The radio-immuno assay (RIA) kits for determination of T₃, T₄, and testosterone were obtained from DSL (Webster, TX, USA). SACcel, the secondary donkey-anti-rabbit antibody complex used in the in-house RIA analyses, was obtained from Welcome Reagents (Beckenham, UK.).

ERG immunohistochemistry was performed as previously described (42 (link)). Brie y, four μm TMA sections were dehydrated and blocked in 0.6% hydrogen peroxide in methanol for 20 min. and were processed for antigen retrieval in EDTA (pH 9.0) for 30 min in a microwave followed by 30 min of cooling in EDTA buffer. Sections were then blocked in 1% horse serum for 40 min and were incubated with the ERG-MAb mouse monoclonal antibody developed at CPDR (9FY, Biocare Medical Inc.) at a dilution of 1:1280 for 60 min at room temperature. Sections were incubated with the biotinylated horse anti-mouse antibody at a dilution of 1:200 (Vector Laboratories) for 30 min followed by treatment with the ABC Kit (Vector Laboratories) for 30 min. The color was developed by VIP (Vector Laboratories,) treatment for 5 minutes, and the sections were counter stained by hematoxylin. ERG expression was reported as positive or negative. ERG protein expression was correlated with clinico-pathologic features.

NeuN immunohistochemistry (Fig 2) was performed using a mouse anti-NeuN antibody (1:2000; Millipore, ref. MAB377) as the primary antibody, and a biotinylated anti-mouse horse antibody (1:500; Vector Laboratories, ref. BA-2001) as the secondary antibody. Briefly, sections were first rinsed three times for 10 min in PBS before being soaked for 1h in 5% normal donkey serum in PBS containing 0.5% Triton X-100. The sections were transferred to the primary anti-NeuN antibody solution for 18h at room temperature, then rinsed three times and soaked in a buffer solution containing the biotinylated secondary antibody for 1h. After three more rinses, the staining was finally amplified using the avidin-biotin peroxidase method (Vectastain ABC kit, Vector Laboratories, Burlingame, CA, USA) and revealed using diaminobenzidine (SK-4100 kit, Vector Laboratories). The second half of the brain sections from the region including the ventral midline thalamus were stained with Cresyl violet (Fig 2). These brain sections were directly collected on slides, contrary to their NeuN counterparts. The successive baths for Cresyl violet staining were as follows: distillated water (1 min); 0.1% Cresyl Violet for (12 min); 70% ethanol (2 min); 95% ethanol (2 min) and 100% ethanol (2 min). Finally, slides were defatted in 100% Xylene (2 times 10 min) before being mounted on DPX medium.