Ab133350

For immunocytochemistry, freshly isolated spermatogonia and round spermatids from P63^(+/−) and wild-type mice were fixed with 4% PFA for 15–30 min, and they were washed three times with cold PBS (Medicago, Uppsala, Sweden) and permeabilized with 0.4% Triton X-100 (Sigma) for 5–15 min. After extensive washes with PBS, these cells were blocked in 5% BSA for 1 h at room temperature and followed by incubation with primary antibodies, including GPR125 (Abcam, ab51705, 1:200), GFRA1 (Abcam, ab8026, 1:200), UCHL1 (AbDSerotec, MCA4750, 1:200), THY1 (Abcam, ab133350 1:200), PRM1 (Santa Cruz Biotechnology, sc-30173, 1:200) overnight at 4 °C. The specificity of these antibodies was evaluated previously and verified to be great^{11 (link)}. After extensive washes with PBS, the cells were incubated with IgGs conjugated with fluorescein isothiocyanate (FITC) (Sigma) or rhodamine-conjugated IgG (Sigma), at a 1:200 dilution for 1 h at room temperature. Replacement of primary antibodies with isotype IgGs in mouse male germ cells served as negative controls. DAPI (4, 6-diamidino-2-phenylindole) was employed to label the cell nuclei, and the images were captured with a Nikon microscope (Tokyo, Japan).