Germ-cell spreads

Chromosome spreads from testes were performed based on a previously published protocol (Peters et al., 1997). Briefly, testes were incubated in a hypotonic extraction buffer pH 8.2 (30 mM Tris-HCL, 50 mM sucrose, 17 mM trisodium citrate dehydrate, 5 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF) for 60 min. Testis tubules were then broken apart in 100 mM sucrose and then spread on a slide dipped in fixation buffer (1% PFA and 0.15% Triton X-100). Slides were incubated overnight in a humidified chamber at room temperature and air-dried. Slides were washed twice in 0.4% Photoflo (Kodak, Rochester, NY) and air-dried.