Zonae pellucidae were removed by a brief incubation with acidic Tyrode’s solution, and zona pellucida-free embryos were washed in phosphate-buffered saline (PBS), fixed in freshly prepared 2% paraformaldehyde for 20 min at room temperature, and washed twice in PBS. Afterward, the embryos were permeabilized in 0.5% Triton X-100 for 25 min, washed three times in PBS, and then blocked with 5% donkey serum for 1 h. Samples were incubated with SIN3A antibody (Cat #3479, Abcam), TRF2 antibody (Cat #ab108997, Abcam), or rabbit polyclonal anti-γH2AX antibody (Cat #80312, Cell Signaling Technology) diluted 1: 100 in blocking solution at 4°C overnight, washed three times, incubated with FITC donkey anti-mouse IgG or FITC anti-rabbit IgG for 1 h at room temperature in the dark, washed again, and finally mounted with VECTASHIELD mounting medium and exposed to 0.5 μg/mL 4, 6-diamidino-2-phenylindole (DAPI, 1 μg/mL, Thermo Fisher Scientific). Images were captured with a ZEISS microscope equipped with fluorescence (Celldiscoverer 7 with LSM900).
Rabbit polyclonal anti γh2ax antibody
Manufactured by Cell Signaling Technology
Sourced in Macao
The Rabbit polyclonal anti-γH2AX antibody is a laboratory reagent used to detect the phosphorylation of the histone variant H2AX, which is an important marker of DNA double-strand breaks. This antibody can be used in various techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to facilitate the study of DNA damage response pathways.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Trf2 antibody, by Abcam (1 mentions)
Vectashield mounting medium, by Thermo Fisher Scientific (1 mentions)
Sin3a antibody, by Abcam (1 mentions)
Phalloidin tritc, by Merck Group (1 mentions)
Fv3000, by Olympus (1 mentions)
2 protocols using rabbit polyclonal anti γh2ax antibody
1
Immunofluorescence Analysis of Zona Pellucida-free Embryos
2
Immunofluorescent Staining of Oocytes
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For immunofluorescent staining, oocytes were fixed in 4% paraformaldehyde in PBS at room temperature for 30 min and then transferred to membrane permeabilization solution (0.5% Triton X‐100) for 20 min. The oocytes were blocked with 1% bovine serum albumin in PBS for 1 h and incubated at 4°C overnight with primary antibody at the concentration indicated in the instructions, specifically, mouse monoclonal anti‐a‐tubulin‐FITC antibody and phalloidin‐TRITC were purchased from Sigma (St. Louis, MO), rabbit polyclonal anti‐Drp1, rabbit polyclonal anti‐γ‐H2AX antibody was purchased from Cell Signaling Technology (Danvers, MA), rabbit polyclonal TPX2 antibody was purchased from Novus Biologicals (Colorado, USA). After washing with 0.1% Tween 20 and 0.01% Triton‐X 100 for 3 times (10 min/time), the oocytes were incubated with secondary antibody for 1 h at room temperature. Finally, the oocytes were stained with Hoechst 33,342 for 10 min. The samples were mounted on glass slides and observed under confocal laser‐scanning microscopy (FV3000, Olympus).
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