Rabbit anti cyp17a1

The Western blots were performed as described previously [36] (link). Briefly, the total protein from the cells was extracted using RIPA buffer. Aliquots of protein were electrophoresed in SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with mouse anti-β-Actin, rabbit anti-Star, goat anti-3β-HSD, rabbit anti-CYP17A1, goat anti-17β-HSD3, rabbit anti-Pcna, rabbit anti-AR, and rabbit anti-ERα (further information on the primary antibodies are shown in Table 1, purchased from Santa Cruz, USA). After washing with TBST buffer, the membrane was then incubated with HRP-labeled goat-anti-mouse IgG, goat-anti-rabbit, and rabbit-anti-goat IgG (all at 1∶2000 dilution, Jackson ImmunoResearch, USA). The final exposure was obtained using enzymatic chemiluminescence (GE Healthcare, USA). The film was then scanned, and the band density was quantified using the ImageJ software, version 1.34 (NIH).

The immunohistochemical analyses were performed as previously described [36] (link). The slides were stained with the following antibodies for chromatic visualization: goat anti-3β-HSD, rabbit anti-CYP17A1 (further information on the primary antibodies is presented in Table 1, purchased from Santa Cruz, USA), biotinylated rabbit-anti-goat, goat-anti-rabbit IgG (all at 1∶200 dilution, Vector Laboratories, USA), and streptavidin-conjugated HRP (1∶200 dilution, Jackson ImmunoResearch, USA). The specific binding was visualized using DAB (1∶20 dilution, Leica, Germany). The sections were counterstained with hematoxylin and mounted for further microscopic analyses. The numbers of positive cells in the testes at PD21.5 and the testes at PD45.5 and PD90.5 were counted in five randomly and ten randomly selected fields, respectively. A total of 5-20 related slides were used to quantify the number of positive cells.