Immunofluorescence Staining of Spermatocytes

Spreads of spermatocytes and immunofluorescence staining were prepared according to the previous references51 (link)52 (link). Briefly, seminiferous tubules were incubated in hypotonic extraction buffer (50 mM Sucrose, 17 mM Sodium citrate, 30 mM Tris (pH 8.2), 2.5 mM DTT, 1 mM PMSF (pH 8.3) and 5 mM EDTA on ice for 20 minutes, minced in 100 mM sucrose, spread on slides and fixed in 1% PFA with 0.1% Triton X-100. Slides were incubated in a humid chamber overnight, dried, and washed in PBS and water containing Photoflo (Kodak, NY, USA). Following blocking in 10% donkey serum and 3% BSA, immunofluorescence staining was performed by incubating with primary antibodies: γH2AX (1:500; abcam) and SYCP3 (1:100; Abcam) overnight at room temperature. Alexa 488 donkey anti-rabbit (1:500, Molecular Probes), Alexa 594 goat anti-mouse (1:200, Molecular Probes) were used as secondary antibodies. Slides were incubated with secondary antibodies at 37 °C for 1 hour in the dark, washed and mounted with Vecta shield cover slips. (Vector Laboratories).