Histone H3 Trimethylation Analysis in Zygotes

Ten hours after the ICSI/ROSI, the zona pellucidae of surviving oocytes were removed using an acetic Tyrode solution, and the naked oocytes were fixed for 30 min at 25 °C in 4% (w/v) paraformaldehyde. The fixed oocytes were washed three times in PBS–polyvinyl alcohol (0.1 mg/mL PVA; Sigma-Aldrich, St. Louis, MO, USA) for 10 min and stored overnight at 4 °C in PBS supplemented with 1% (w/v) bovine serum albumin (BSA/PBS, Sigma-Aldrich) and 0.1% (v/v) Triton X-100 (Nacalai Tesque, Inc., Kyoto, Japan). The following procedure was previously described in^{7 (link)}. The primary antibodies used were an anti-histone H3 (trimethyl K9) mouse monoclonal antibody (1:500; Abcam, Cambridge, UK) or an anti-pan-histone mouse monoclonal antibody (1:500: Merck, Darmstadt, Germany). The secondary antibodies used were Alexa Fluor 488-labeled goat anti-mouse IgG (1:500; Molecular Probes, Eugene, OR, USA) and Alexa Fluor 568-labeled goat anti-rabbit IgG (1:500 dilution; Molecular Probes). DNA was stained with 4′6-Diamidino-2-phenylindole (DAPI; 2 μg/mL; Molecular Probes). The brightness of the whole male pronucleus was measured using ImageJ and was then subtracted from the brightness of the zygote cytoplasm.