Anti phospho histone h3 antibody

Germline immunostaining was performed as described by Navarro et al. (2001) (link). Anti-Phospho-Histone H3 antibody was applied at a 1:300 dilution (Abcam, Cambridge, England) and a secondary antibody and DAPI were applied at 1:1000 (Thermo Scientific, MA, USA). Slides were examined using an Olympus IX81microscope attached to an X-Cite series 120Q fluorescent light box.

After indicated conditions with ARN‐3236, cells were pelleted and fixed with 70% ethanol at 4 °C overnight. For cell cycle analysis, FxCycle PI/RNase staining solution (Invitrogen) was used to stain the cells at 4 °C overnight. For phospho‐H3 staining, cell pellets were washed with PBS once and resuspended in PBS containing 0.25% Triton X‐100 on ice for 15 min. The pellets were then stained with rabbit anti‐phospho‐Histone H3 antibody (1 : 1000; Abcam) in 1% BSA at 4 °C overnight. After washing with PBS, the cell pellets were probed with anti‐rabbit Alexa Fluor 488 secondary antibody (Invitrogen) for 1 h. The pellets were counterstained with propidium iodide (25 μg·mL⁻¹) before analysis by flow cytometry (FACSCanto II; BD Biosciences, Franklin Lakes, NJ, USA).