Rabbit anti phospho histone3

Cells were rinsed once with phosphate-buffered saline (PBS, pH 7.4) and then fixed in 4% paraformaldehyde for 15 min. After washed with 1X PBT (PBS with 0.2% Triton X-100), cells were incubated in goat serum blocking buffer for one hour prior to incubation with primary antibodies of mouse anti-TUJ1 (1:1000; Covance), mouse anti-MAP2 (1:500; Millipore), rabbit anti-Caspase3 (1:200; Cell Signaling), rabbit anti-phospho-Histone3 (1:0000; Abcam) and rabbit anti-GFP (1:1000; Invitrogen) at 4 °C for 24 hours. Labeling was visualized with DyLight^TM 550- or 488-conjugated goat anti-mouse or anti-rabbit IgG secondary antibody (1:1000; Abcam) at room temperature for two hours. After wash, cell nuclei were stained by DAPI (Invitrogen) for 30 min and cells were preserved in PBS containing 0.02% sodium azide or mounted with anti-fade media (Pro-Long Gold, Invitrogen). For BrdU incorporation, 5 µM of BrdU was added into the media two hours before fixation. Cells were fixed and treated with 2 N HCl at 37 °C for 30 min and 0.1 M of sodium borate for 10 min at room temperature before blocking. Cells were then incubated with rat anti-BrdU (1:500; Accurate) at 4 °C for 24 hours. Labeling was visualized with DyLight^TM 488-conjugated goat anti-rat IgG secondary antibody (1:1000; Abcam).

The following primary antibodies were used for this study: rat anti-α-Tub (Serotec; 1:500; available from Bio-Rad; Cat#MCA77G), mouse anti-α-Tub (DM1A, Sigma; 1:2500; Cat# T9026), rabbit anti-phospho-Histone 3 (Abcam; 1:1000; Cat# sc-56739), chicken anti-GFP (Abcam; 1:1000; Cat# ab13970), rabbit anti-Ran (Abcam; 1:100; Cat# ab11693), rat anti-Miranda (1:400; gift from Chris Doe), mouse anti-aPKCζ (Santa Cruz; 1:50; discontinued) and guinea pig anti-Sqh1P (1:300)^{66 (link)}. Secondary antibodies were from Molecular Probes and the Jackson Immuno laboratory.