P450scc

Immunofluorescence staining was performed on paraffin sections. After quenching the endogenous peroxidases with 3% H₂O₂ in 10% methanol, targeted antigens were retrieved by boiling the slides in an antigen-unmasking solution (H-3300, Vector Laboratories). Sections were treated with blocking reagent (all supplied by Roche) before application of primary antibodies for Sertoli and Leydig cell detection: GATA-1 (1:200, Cell Signaling Technology), and P450scc (1:100, Cell Signaling Technology), respectively. The number of Sertoli cells were quantified per seminiferous tubule by averaging the number of positive GATA-1 signals for 20 tubules within each testes (n = 5). Leydig cell signals were quantified via ImageJ software by measuring fluorescence intensities of interstitial space P450scc signals and normalized to background (n = 6). Cell proliferation was assayed by immunofluorescence staining using Ki-67 antibodies (1:100, Thermo Scientific) and apoptosis was examined using immunofluorescence staining of caspase-3 antibody (1:200, Cell Signaling Technology). Dhh expression was analyzed using Dhh antibody (1:100, Santa Cruz Biotechnology Inc.). MVH staining (1:200, ab13840) was performed in order to stain spermatogonium. All primary antibodies were incubated at 4°C overnight.