Rabbit anti phospho histone h2a x

Oocytes were fixed in 3.7% paraformaldehyde dissolved in polyvinyl alcohol in PBS (PVA-PBS) for 30 min at room temperature or overnight at 4°C. For permeabilization, fixed oocytes were transferred into 0.5% Triton X-100 for 1 h at room temperature. After blocking in 1% BSA in PBS for 1 h, oocytes were stained with the primary antibodies rabbit anti-phospho-Histone H2A.X (1:200; #9718T; Cell Signaling Technology, Danvers, MA) and mouse anti-pericentrin (1:200; #611814; BD Biosciences, Franklin Lakes, NJ) at 4°C overnight. After washing the oocytes three times with PVA-PBS, they were labeled with secondary antibodies Alexa Fluor 594 goat anti-rabbit and Alexa Fluor 488 goat anti-mouse (1:200; #A11012; #A11001, respectively; Thermo Fisher Scientific) at room temperature for 2 h. For spindle staining, the oocytes were incubated with mouse monoclonal FITC-conjugated anti-α-tubulin antibody (1:200; Sigma-Aldrich) for 2 h at room temperature. Stained oocytes were washed extensively with PVA-PBS and incubated with Hoechst 33342 (10 μg/mL; #B2261; Sigma-Aldrich) for 20 min. After washing, the samples were mounted on glass slides in Vectashield containing DAPI (Vector Laboratories, Burlingame, CA) and observed under a fluorescence microscope (Zeiss, Oberkochen, Germany).