Mca2336

Embryonic gonads were collected from E12.5 to E18.5. Gonads were dissected in PBS and fixed in 4% PFA for up to 24 h, stored in 70% ethanol and embedded in paraffin. Tissue sections (5 µm) were cut and mounted on glass slides. Immunofluorescence and immunohistochemistry of deparaffinized sections were performed as described previously (Chen et al. 2013) (link). Primary antibodies included anti-EMX2 (ab110112, 1:200; Abcam), anti-DDX4 antibody (ab13840, 1:200; Abcam), anti-KI67 (ab15580, 1:400; Abcam), anti-STELLA (sc-67249; Santa Cruz), anti-SOX9 (ab3697, 1:400; Abcam), anti-STRA8 (ab49602; Abcam) and anti-DAZL (MCA2336, 1:200; AbD Serotec (San Diego, CA, USA)). FITC or TRITC-conjugated secondary antibodies were from Jackson ImmunoResearch. The sections were counterstained with DAPI (Sigma-Aldrich) to label the nuclei. To determine the percentage of gonocytes that were positive for KI67, we counted at least ten sections for each group.

After rehydration and antigen retrieval, the 5-μm sections were incubated with 5% donkey serum in 0.3% Triton X-100 for 1 h. Then, the sections were incubated with the primary antibodies for 1.5 h, and the corresponding fluorescein isothiocyanate-conjugated donkey anti-rabbit immunoglobulin G (IgG; 1:150; Jackson Laboratory) and TRITC-conjugated donkey anti-mouse IgG (1:150; Jackson Laboratory) for 1.5 h at room temperature. The following dilutions of primary antibodies were used: STELLA (1:200; sc-67249; Santa Cruz Biotechnology), OCT4 (1:300; Sc-8628; Santa Cruz Biotechnology), WT1 (1:200; 2797-1; Epitomics), MVH (1:500; ab13840; Abcam), PRMT5 (1:200; 07-405; Millipore), DAZL (1:100; MCA2336; AbD Serotec), STRA8 (1:200; ab49405; Abcam), SCP3 (1:200; ab15093; Abcam), γH2AX (1:400; 05-636; Millipore), Ki-67 (1:200; ab15580; Abcam) , H3R2me2s (1:50; ABE460; Millipore), and H4R3me2s (1:100; ab5823; Abcam). After three washes in PBS, the nuclei were stained with 4′,6-diamidino-2-phenylindole. The sections were examined using a confocal laser scanning microscope (Carl Zeiss Inc.).