Immunofluorescence staining

Testes were dissected in PBS, fixed in 4% formaldehyde for 20mins and washed by PBSTw (PBS with 0.1% Tween-20) for 3 times. Permeabilization of testes was done by incubation with PBST (PBS with 0.5% Triton-X) for 30mins. Testes were then blocked by 5% BSA in PBSTw for at least an hour, before incubation with primary antibody in 5% BSA in PBSTw at 4°C overnight. Testes were washed 3 times with PBSTw and incubated with secondary antibody in 5% BSA in PBSTw at room temperature for 2hrs, followed by another 3 washes with PBSTw. Before mounting in VECTA-SHIELD, testes were stained by DAPI (1:5000) for 10mins and rinsed once with PBS. The following primary antibodies were used: mouse anti-Fas3 (7G10, 1:200), mouse anti-γH2Av (UNC93-5.2.1, 1:400) and rat anti-Vasa (concentrated, 1:100) were obtained from Developmental Studies Hybridoma Bank.