Anti phospho histone3

Under different experimental conditions, hESCs were seeded onto four chambered glass slides. Paraformaldehyde (PFA, 4%) in PBS was used for fixation, permeabilization for intracellular markers was achieved with 0.2% Triton X-100 in PBS and blocked with normal goat serum. Fixed cells were incubated with primary antibodies: CDK2AP1 (Santa Cruz, CA, USA) and Anti-Phospho-Histone-3 (Cell Signaling, MA, USA). Goat anti-rabbit IgG conjugated to Alexa 594 (Invitrogen, CA,) was used as a secondary antibody. Fluorescent images were acquired using a Cool- Snap EZ camera (Photometrics, Tucson, AZ) mounted on a Nikon Eclipse TE 2000-S inverted microscope (Nikon, Melville, NY) with attached image analysis software. All image settings were controlled for uniform acquisition between samples. Specifically, uniform exposure time was maintained for images acquired from experimental samples as well as negative controls for background subtraction. For experiments involving determination of mitotic index based on phospho-histone 3 staining, random fields were selected and counting was performed in triplicate by counting around 500 cells during each analyses. Mitotic index was calculated by dividing the number of phospho-histone 3 positive cells, over the total number of cells.