Anti 3β hsd

Perfused testes were assembled in five samples per time point in a tissue array and paraffin embedded and 6-μm thick cross sections were cut, mounted, and dewaxed. The immunohistochemical staining for Leydig cell specific protein CYP11A1, 3β-HSD1 and 11β-HSD1 was performed according to the previous method.16 (link)17 (link)18 (link) In brief, the sections were incubated in primary antibody polyclonal rabbit anti-CYP11A1 (RDI Research Diagnostics, Inc., Flanders, NJ, USA; dilution 1:1000), anti-3β-HSD (Abcam, CA, USA, dilution1:1000) and anti-11β-HSD1 (inhouse made, dilution 1:1000), at 4°C overnight. The following morning the sections were washed in Tris-buffer for 5 min; incubated in secondary goat anti-rabbit antibody (Santa Cruz Biotech, CA, USA) 1:1000 diluted in Tris-buffer supplemented for 30 min; washed in Tris-buffer for 5 min and incubated with DAB/H₂O₂ solution for 10 min. They were then washed for 5 min in distilled H₂O and subsequently dehydrated in serial isopropyl alcohol baths and cover-slipped. In control experiments, sections were incubated with nonimmune rabbit IgG (CYP11A1, 3β-HSD1 and 11β-HSD1).