Immunofluorescence Staining of Embryonic Zygotes

Immunofluorescence staining was performed as previously described (Qian et al., 2016 (link); Zhang et al., 2016 (link)). Zygotes were digested with Tyrode’s acid solution (Sigma, T1788) for 30 s to remove the zonae pellucidae, before being fixed in 4% paraformaldehyde for 30 min, and mounted on a polylysine-coated slide. Then, the zygotes were permeabilized with 0.5% Triton X-100 for 20 min, and blocked with PBS containing 3% BSA and 10% goat or donkey serum for 1 h. After that, samples were immunolabeled with primary antibody at 4°C overnight, followed by Alexa 488 (goat anti-rabbit, 1:400, Abcam, ab150077) or Cy 3 (goat anti-rabbit, 1:400, Abcam, ab6939) IgG secondary antibody for 1 h; the nuclei were stained with DAPI (Biosharp, Beijing, China) for 20 min. Quantification of fluorescence signal was performed with ImageJ software. Primary antibodies: rabbit anti-phosphor-AMPK (α, phospho Thr172; 1:200, Abcam, ab23875), rabbit anti-phospho-histone H2AX (phospho Ser139; 1:200, Abcam, ab2893), rabbit anti-phospho-histone H3 (phospho Ser10; 1:600, Cell Signaling Technology, #53348), and rabbit anti-p21 (1:100, Affinity, AF6290).