Anti histone h3 dimethyl k9 mouse monoclonal antibody

Ten hours after the ICSI, the zona pellucidae of surviving oocytes were removed using acetic tyrode solution, and then the naked oocytes were fixed for 30 min at 25°C in 4% (w/v) paraformaldehyde (PFA). The fixed oocytes were washed three times in phosphate-buffered saline (PBS)–polyvinyl alcohol (0.1 mg/ml; Sigma-Aldrich, St. Louis, MO) 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 procedures are previously described in (30 (link)). The primary antibodies used were an anti–phospho-H2AX (Ser¹³⁹) rabbit polyclonal antibody (1:500; Millipore-Merck, Darmstadt, Germany) and an anti-histone H3 (dimethyl K9) mouse monoclonal antibody (1:500, Abcam, Cambridge, UK). The secondary antibodies used were Alexa Fluor 488–labeled goat anti-mouse immunoglobulin G (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.

Histone H2Ax is one of the H2A variants. The serine at position 139 of H2Ax is rapidly phosphorylated within seconds of DNA damage. The phosphorylated form of H2Ax, designated as
gamma-H2Ax, forms foci at sites of DNA damage, which leads to the recruitment of various repair and cell-cycle checkpoint proteins [21 (link)]. Therefore,
gamma-H2Ax foci formation was used as a marker of DNA double-strand breaks in male and female pronuclei, and histone H3K9 me2 signals were used to distinguish female and male pronuclei. All
specimens were fixed 10 h after ICSI with 4% paraformaldehyde (PFA; Wako Pure Chemical, Osaka, Japan) containing 0.2% Triton X at RT for 20 min and stored in a refrigerator until staining.
Primary antibodies used for the immunostaining of zygotes included the anti-phospho-H2Ax (Ser139) rabbit polyclonal antibody (1:500; Millipore-Merck, Darmstadt, Germany) and anti-histone H3
(dimethyl K9) mouse monoclonal antibody (1:500; Abcam, Cambridge, UK). 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; Molecular Probes). DNA was stained with 4′6-diamidino-2-phenylindole (2 µg/ml; Molecular Probes). The brightness of each male
pronucleus was measured using ImageJ software and was subtracted from the brightness of the zygote cytoplasm.