Immunohistochemical Analysis of Genital Ridge

For immunohistochemical analyses, genital ridges isolated from embryos (E10.5–14.5) were fixed in 4% paraformaldehyde, embedded in OCT compound, and then frozen. Sections prepared from the frozen tissues were mounted on glass slides and subjected to immunostaining as described previously^{25 (link)}. The primary antibodies used were as follows: anti-GENA (TRA98; BioAcademia, 73–003), anti-MAX (Santa Cruz Biotechnology, sc-197), anti-OCT4 (Santa Cruz Biotechnology, sc5279), and anti-cPARP (BD Pharmingen, 558,710). The anti-GENA antibody was used at 1:400 dilution and the other antibodies were used at 1:100 dilution. Immunostained tissues/cells were observed under a confocal laser scanning microscope (TCS SP8; Leica Microsystems, Wetzlar, Germany). The average staining intensities of MAX and SYCP3 in somatic cells were arbitrarily set to 100.