Sc 30820

IF was performed using both paraffin and frozen sections, and IHC using paraffin sections. Testes were obtained immediately following euthanasia by CO₂ asphyxiation, fixed in 4% paraformaldehyde, embedded in paraffin and obtained paraffin sections at 5 μm with a microtome. Frozen sections at 8 μm obtained in a cryostat at -22°C were fixed in 4% PFA for 10 min. IF was performed using either the FITC or TRITC-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA). For IHC, tissue sections were de-paraffinized and rehydrated, to be followed by antigen retrieval in 10 mM sodium citrate buffer for 15 min. Positive staining was visualized using DAB substrate kits (Zhong Shan Technology, China) and sections were counterstained with hematoxylin. Antibodies were obtained commercially as follows: Ki67 (1:1000, ab15580, Abcam), α-SMA (1:400, S0010/ab137734, Epitomics/Abcam), PCNA (1:100, 2586, Cell Signaling Technology), HSD3B1 (1:400, sc-30820, Santa Cruz), CYP11A1 (1:500, AB1244, Chemicon/Millipore), VCAM1 (1:400; AF643; R&D) and JAG1 (1:50, sc-6011, Santa Cruz). IF and IHC was performed as described [15 (link), 16 (link)]. Images were examined and acquired using a Nikon Eclipse 80i fluorescence microscope (Tokyo, Japan) with a built-in Nikon CCD camera for image acquisition. Image overlays and relative fluorescence intensity was quantified using Image J software.

Cells were washed with PBS and then fixed with 4% paraformaldehyde (Biosesang, Gyeonggi-do, Korea) for 20 min at room temperature (RT). Fixed cells were permeabilized with 0.1% Triton X-100 (Sigma) in PBS for 10 min. After permeabilization, the cells were washed twice with PBS, blocked with blocking solution (DAKO North America Inc., Carpinteria, CA, USA) for 1 h at RT, and incubated with primary antibodies overnight at 4 °C. On day 2, cells were washed twice with PBS, incubated with secondary antibodies for 1 h at RT, and then analyzed. The following antibodies were used: SF1 (1:100; LS-C162999, Lifespan Biosciences, Inc., Seattle, WA, USA), GATA4 (1:100; sc-25310, Santa Cruz Biotechnology, Santa Cruz, CA, USA); 3βHSD (1:50; sc-30820, Santa Cruz), and LHCGR (1:100; ab204950, Abcam, Cambridge, MA, USA). The secondary antibodies used were Alexa Fluor 488-conjugated anti-rabbit IgG (1:200, Invitrogen, Carlsbad, CA, USA) for SF1, Alexa Fluor 555-conjugated anti-mouse IgG (1:200, Invitrogen) for GATA4 and LHCGR, and Alexa Fluor 555-conjugated anti-goat IgG (1:100, Invitrogen) for 3βHSD.