Af1446

The total expression of AMH and superoxide dismutase 2 (SOD2) was analyzed in the collected ovarian tissue. The proteins were extracted from ten ovaries per group. Tissue extracts (30 μg) were electrophoresed on a 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. The membranes were blocked using 5% nonfat milk in Tris-buffered saline (10 mM Tris and 150 mM phosphate buffered saline, pH 8.0) for 1 h at room temperature. Next, the membranes were incubated overnight at 4°C with diluted primary antibodies (AMH, AF1446, 1:250 dilution, goat antibody, R&D Systems, Minneapolis, USA; SOD2, ab68155, 1:1000 dilution, rabbit monoclonal antibody, Epitomics, California, USA; and actin, BM0005, 1:1000 dilution, rabbit polyclonal antibody, Boster, China). The blots were subsequently incubated with secondary antibodies (1:1000 dilution). The immunoreactive bands were observed using alkaline phosphatase (ALP) and BCIP/NBT staining. Each blot was completed in quadruplicate.

Mouse embryos were fixed in 4% PFA in PBS at 4°C, embedded in paraffin, sectioned at 5μm, and immunofluorescence performed as described previously [39 (link)]. Primary antibodies used for this study were anti-HMGCS2 rabbit monoclonal (1:50; ab137043, Abcam), anti-SOX9 sheep polyclonal (1:100; [40 (link)]), anti-MVH goat polyclonal (1:200; AF2030, R&D systems), anti-AMH goat polyclonal (1:200; sc6886, Santa Cruz), anti-AMH goat polyclonal (1:50; AF1446 R&D systems), anti-SYCP3 mouse monoclonal (1:100; ab97672, Abcam), anti-FOXL2 rabbit polyclonal (1:300; [41 (link)]) and anti-CYP11A1 rabbit polyclonal [42 (link)]. Secondary antibodies used were donkey anti-rabbit Alexa 488, donkey anti-rabbit Alexa 568, donkey anti-goat Alexa 488, donkey anti-goat Alexa 546, donkey anti-mouse Alexa 488, and donkey anti-sheep Alexa 647 obtained from Invitrogen and used at 1:300. Images were taken with a Zeiss LSM 510 Meta confocal microscope at the Australian Cancer Research Foundation Dynamic Imaging Centre for Cancer Biology, University of Queensland and with a Zeiss LSM800 confocal microscope at the Biological Optical Microscopy Platform (BOMP) at the Department of Anatomy and Neuroscience, The University of Melbourne.