Sin3a antibody

Zonae pellucidae were removed by a brief incubation with acidic Tyrode’s solution, and zona pellucida-free embryos were washed in phosphate-buffered saline (PBS), fixed in freshly prepared 2% paraformaldehyde for 20 min at room temperature, and washed twice in PBS. Afterward, the embryos were permeabilized in 0.5% Triton X-100 for 25 min, washed three times in PBS, and then blocked with 5% donkey serum for 1 h. Samples were incubated with SIN3A antibody (Cat #3479, Abcam), TRF2 antibody (Cat #ab108997, Abcam), or rabbit polyclonal anti-γH2AX antibody (Cat #80312, Cell Signaling Technology) diluted 1: 100 in blocking solution at 4°C overnight, washed three times, incubated with FITC donkey anti-mouse IgG or FITC anti-rabbit IgG for 1 h at room temperature in the dark, washed again, and finally mounted with VECTASHIELD mounting medium and exposed to 0.5 μg/mL 4, 6-diamidino-2-phenylindole (DAPI, 1 μg/mL, Thermo Fisher Scientific). Images were captured with a ZEISS microscope equipped with fluorescence (Celldiscoverer 7 with LSM900).