Zeocin is a DNA DSBs reagent. Several concentration gradients were set up to investigate the effects of Zeocin on DNA DSBs in oocytes. The bovine COCs collected were cultured in mature culture medium with 0, 10, 50, 351.68 or 1758.38 μM Zeocin (Figure S2B ) for 2 h, in order to induce DNA DSBs, which were detected by immunofluorescence staining for γH2AX.
Immunofluorescence reagents were all purchased from Beyotime Biotechnology (Beyotime Biotechnology, Shanghai, China). To label the γH2AX, briefly [62 (link),65 (link)], oocytes were fixed in 4% paraformaldehyde (PFA) at room temperature for 40 min, followed by permeabilization in 0.5% Triton X-100 for 20 min. After blocking in 1% BSA for 1 h, the oocytes were incubated with γH2AX antibody (Abcom, Shanghai, China) at 4 °C overnight, washed three times and incubated with the second antibody at room temperature for 1 h. Then, the oocytes were stained with Hoechst 33,342 (10 μg/mL) for 5 min. Finally, the oocytes were observed under a fluorescence inversion microscope (Axiocam MRm; Carl Zeiss, Shanghai, China).
Immunofluorescence reagents were all purchased from Beyotime Biotechnology (Beyotime Biotechnology, Shanghai, China). To label the γH2AX, briefly [62 (link),65 (link)], oocytes were fixed in 4% paraformaldehyde (PFA) at room temperature for 40 min, followed by permeabilization in 0.5% Triton X-100 for 20 min. After blocking in 1% BSA for 1 h, the oocytes were incubated with γH2AX antibody (Abcom, Shanghai, China) at 4 °C overnight, washed three times and incubated with the second antibody at room temperature for 1 h. Then, the oocytes were stained with Hoechst 33,342 (10 μg/mL) for 5 min. Finally, the oocytes were observed under a fluorescence inversion microscope (Axiocam MRm; Carl Zeiss, Shanghai, China).