Sc 46081

For histology testes were fixed for 6 hours in Bouin’s solution and then embedded in Technovit 7100 resin. Sections (2.5μm) were cut and stained with H&E. Immunohistochemistry was carried out as described [11 (link),12 (link)]; testes were embedded in paraffin and sections (5μm) prepared and stained using anti-CYP11A1 (rabbit polyclonal, donated by Dr AH Payne, Stanford University) diluted 1/500 or anti-CYP17A1 (sc-46081 Santa Cruz, lot number F2014, Heidelberg, Germany) diluted 1/400 and using the appropriate secondary antibodies conjugated to biotin, diluted at 1/500 in the blocking serum. This step was followed by incubation with horseradish peroxidase labelled avidin-biotin complex (VectorLabs, Peterborough, UK). The revelation was undertaken with diaminobenzidine DAB (Immpact DAB;VectorLabs, Peterborough, UK). Slides were counterstained with haematoxylin, dehydrated and mounted with Pertex mounting medium (Cell Path, Hemel Hempstead, UK). Cell numbers were measured by the optical dissector as previously shown [8 (link), 9 (link)]. The cells were recognised by their position, round nucleus and relatively abundant cytoplasm. The total testis volume was estimated using the Cavalieri principle.

Embryos, gonadal and aorta tissue had been fixed in 4% paraformaldehyde and embedded in paraffin. Slices were pretreated with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20) for epitope demasking and 0.3% hydrogen peroxide to reduce non-specific background due to endogenous peroxidase. CYP17A1 was detected using goat polyclonal antibody sc-46081 (Santa Cruz Biotechnology, Inc) at a dilution of 1:50 and an anti-goat biotinylated secondary antibody (Vectastain BA-5000, Vector Laboratories Inc., Burlingame, CA, USA). Bound antibodies were visualized using the ABC kit (Vectastain, PK-4000) and DAB 2 component chromogen kit (DCS. Hamburg, Germany) or using using donkey anti-goat AP antibody (Supplementary Fig. 2a,b).