Vecta shield cover slips

Spreads of spermatocytes and immunofluorescence staining were prepared according to the previous references^{38 (link),39 (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 dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (pH 8.3), and 5 mM EDTA) on ice for 20 min, 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 phosphate-buffered saline (PBS) and water containing Photoflo (Kodak, NY, USA). Following blocking in 10% donkey serum and 3% bovine serum albumin, immunofluorescence staining was performed by incubating with the primary antibodies: γH2AX (1:1000; Novus, Cat# NB100–384), SYCP3 (1:100; Abcam, Cat# ab97672 or Novus, Cat# NB300–232), DMC1 (1:100, Santa Cruz, Cat# sc-22768), MLH1 (1:100, BD, Cat# 551092), SYCP1 (1:100, NOVAS, Cat# NB300–229), and RNA-Polymerase II (1:200, Active Motif, Cat# 39097), overnight at room temperature. Alexa 488 (1:400, Thermo Fisher) or Alexa 555 (1:200, Thermo Fisher) fluorescent secondary antibody was used. Slides were incubated with secondary antibodies at 37 °C for 1 h in dark, washed, and mounted with Vecta shield cover slips (Vector Laboratories, Cat# H-1000).