Immunostaining of nuclei spreads and fixed nuclei

Characterization of Hells cKO spermatocytes and meiotic staging of spermatocytes after synchronization were performed on nuclei spreads. Meiotic staging after Fluorescence-Activated Cell Sorting (FACS) was performed using fixed nuclei deposited on poly-lysine coated slides. Spreads were prepared with the dry down technique, as described (Peters et al., 1997), and immunostaining was performed as described (Grey et al., 2009). Staging criteria were the following: pre-leptotene nuclei had weak SYCP3 nuclear signal and no or very weak γH2AFX signal; leptotene nuclei were γH2AFX-positive and SYCP1-negative; early/mid zygotene nuclei had less than nine fully synapsed chromosomes; late zygotene had nine or more fully synapsed chromosomes; and pachytene cells had all chromosomes fully synapsed, excepted for the sex chromosomes. The following antibodies were used: rabbit anti-PRDM9 (Grey et al., 2017), (1:200), rabbit anti-HELLS (NB100-278, Novus, 1:200), mouse anti-HELLS (sc46665, Santa Cruz, 1:100), rabbit anti-DMC1 (H-100, Santa Cruz, 1:200), guinea-pig anti-SYCP3 (Grey et al., 2009, 1:500), anti-SYCP1 (ab15090, Abcam, 1:400) and anti-γH2AFX (05–636, Millipore, 1:10,000).