Alexa fluor 568 labeled goat anti rabbit igg

Primary cultured astrocytes grown on coverslips were incubated with recombinant mouse OPN (1 mg/mL) or recombinant human TNF-α (50 ng/mL: Pepro Tech) for 6 h at 37 °C under a 5% CO₂/95% air atmosphere. To detect actin, the cells were washed with PBS, fixed in 4% PFA/0.1 M PB (pH 7.4) for 15 min at room temperature, and treated for 15 min with PBS containing 0.2% Triton X-100. G- and F-actin were then labeled using Alexa488-conjugated deoxyribonuclease I (1:500, Molecular Probes) and rohdamine-conjugated phalloidin (1:1,000, Invitrogen), respectively. For phospho-ERM labeling, the cells were washed with PBS containing 30 mM glycine (G-PBS), fixed with ice-cold 10% trichloroacetic acid for 15 min, and treated for 15 min with G-PBS containing 0.2% Triton X-100. After blocking the cells for 1 h in G-PBS containing 2% BSA and 3% normal goat serum, they were incubated for an additional 1 h with rabbit anti-phospho-ERM (1:400, Cell Signaling Technology). The immune-signals were then visualized using Alexa Fluor568-labeled goat anti-rabbit IgG (1:400, Molecular Probe), and nuclei were visualized using DAPI (1:1,000, Molecular Probe). For Western blot analyses, cell lysates were prepared in RIPA buffer containing 1x protease inhibitor cocktail as described above, and were probed with rabbit anti-ERM (1:1,000, Cell Signaling Technology) or anti-phospho-ERM.

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.

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 recruits various repair and cell-cycle checkpoint proteins^{35 (link)}. Therefore, gamma-H2AX foci formation was used as a marker of DNA double-strand breaks in male and female pronuclei, and histone H3K9me2 signals were used to distinguish female pronuclei from male pronuclei. All specimens were fixed 10 h after ICSI and stored in refrigerator until staining. Primary antibodies used for immunostaining 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 (DAPI, 2 μg/ml; Molecular Probes). The brightness of each male pronucleus was measured using ImageJ software and subtracted from the brightness of the zygote cytoplasm.

Blastocysts were used for the immunofluorescence staining applied to evaluate the quality of the embryos [32 (link)]. Following three washes in PBS containing 0.1% polyvinyl alcohol (PBS-PVA), blastocysts were fixed for 60 min in PBS-PVA containing freshly prepared 2% (w/v) paraformaldehyde and 0.2% (v/v) Triton X at 24 °C. After fixation, oocytes were washed with PBS-PVA (three times for 15 min each) and oocytes were immersed in PBS-PVA containing 2.5% (v/v) Tween 20 (GE Healthcare Co., Chicago, IL, USA) for 2 min at 24 °C. Blastocysts were washed with PBS-PVA (three times for 15 min each) again, incubated for 2 h in PBS-PVA containing 1% (w/v) BSA (PBS-BSA) at 4 °C and thereafter in blocking buffer (PBS-BSA containing 10% (v/v) goat serum) for 40 min at 24 °C. The blastocysts were incubated in primary antibody overnight at 4 °C. The primary antibodies used were an anti-CDX2 rabbit monoclonal antibody (1:500; BioGenex, Fremont, CA, USA) to detect the TE cells and an anti-NANOG mouse polyclonal antibody (1:500; Abcam, Cambridge, UK) to detect the ICM cells. Alexa Fluor 568-labeled goat anti-rabbit IgG and Alexa Fluor 488-labeled goat anti-mouse IgG (both 1:500; Thermo Fisher Scientific, Waltham, MA, USA) were used as secondary antibodies to detect the ICM and TE cells, respectively. DNA was stained with DAPI (2 μg/mL; Molecular Probes, Eugene, OR, USA).