D2c8 xp rabbit mab

Cell proliferation was assessed using the pHH3 (Ser10) (D2C8) XP^® Rabbit mAb (Cell Signaling Technology). Embryos were fixed at 36 hpf in 4% PFA overnight at 4°C with gentle agitation. Embryos were dehydrated and rehydrated with serial dilutions of MeOH and PBTx at room temperature. Embryos were washed 4 times for 30 min each in IB buffer (1% BSA, 0.5% TritonX100, and 1% DMSO in 1X PBS). Embryos were incubated with IB containing 5% Normal goat serum (NGS) for 30 min. Embryos were then incubated with the primary antibody in IB + 5% NGS at a 1:250 dilution overnight at 4°C with gentle agitation. Embryos were washed with IB once for 30 min, then with IB +5% NGS for 30 min. Embryos were incubated with the Invitrogen Goat anti-Rabbit IgG (H + L) Secondary Antibody, Alexa Fluor™ 568 in IB + 5% NGS at a 1:500 dilution overnight at 4°C with gentle agitation. Embryos were washed 3 times with PBTx for 15 min each and postfixed in 4% PFA for 30 min. Embryos were washed 3 times with 1X PBS for 5 min each and stored in 1X PBS for imaging.

HeLa cells were seeded and treated as described for cell cycle analysis. Staining was performed as described for cell cycle analysis, after which cells were permeabilized using 80% ice cold methanol at minus 20 °C for 10 min. Phospho-Histone H3 (Ser10) (D2C8) XP^® Rabbit mAb (Cell Signaling Technology, Danvers, MA, USA) was used to determine the presence of phosphorylated Histone H3. After permeabilization the cells were blocked using PBS containing 0.5% BSA and thereafter incubated with the primary antibody (1:250) for 1 h at 37 °C. Cells were washed and incubated with a conjugated secondary antibody (1:1000), anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor^® 488 Conjugate) (Cell Signaling Technology, Danvers, Massachusetts, USA) for 30 min at 37 °C in the dark. After staining with the secondary antibody, the cells were washed once to eliminate unbound antibodies and then imaged.