The spermatocyte spreads and seminiferous tubules squashes were blocked for 30 min with 1% BSA and 0.05% Tween in PBS (PBS-T) and then were incubated overnight at 4 °C with the following primary antibodies: Sycp3 (Abcam, ab15093 and ab97672, 1:800; and Santa Cruz, sc-20845, 1:20); Sycp1 (Abcam, ab15087, 1:800) Dmc1 (Santa Cruz, sc-22768, 1:50); Trf1 (Alpha Diagnostics, TRF12-S, 1:50); Sun1 (Abcam, ab103021, 1:50); H1t (gift from Dr Mary Ann Handel, The Jackson Laboratory, Bar Harbor, Maine, USA, 1:200) H3K9-3me (Milipore, 05-1250, 1:2000); γH2AX (Milipore, 05-636, 1:300); Mlh1 (Becton Dickinson, 551091, 1:100); Rad51 (Santa Cruz, sc-8349, 1:100) Terb-1 (affinity-purified rabbit antiserum, 1:100)34 (link) and Cdk2 (Abcam, ab7954, 1:50). RingoA was detected using the R55A mouse monoclonal antibody12 (link), which recognizes the sequence NDHQSNK corresponding to amino acids 283-289 (hybridoma supernatant, 1:1). The slides were then washed in blocking buffer and incubated for 1 h at 37 °C with appropriate Alexa Fluor-labelled secondary antibodies (Invitrogen, A21442, A31571, A21441, A21468, A21203, A11017, 1:300). Finally, the slides were washed and mounted in ProLong Gold antifade reagent (Molecular Probes).
For telomere FISH staining, Telomere PNA FISH kit/cy3 (Dako, K5326) was used on 16 dpp testis spreads following the protocol described by the manufacturer.
For telomere FISH staining, Telomere PNA FISH kit/cy3 (Dako, K5326) was used on 16 dpp testis spreads following the protocol described by the manufacturer.