Anti histone h2a x

For whole mount immunofluorescence on WT and zep mutants, embryos were fixed overnight and washed in phosphate buffered saline (PBS) and then molecular grade water. Embryos were dehydrated in 100% methanol and placed at -20°C for at least 1 hour. The embryos were then stepwise rehydrated and blocked for 1 hour at room temperature (0.1 g BSA, 100 μl Triton x-100, 100 μl Tween-20, 100 μl DMSO, 500 μl fetal calf serum (FCS) filled to 10 ml with 1x PBS). Directly following blocking, primary antibodies were incubated at 4°C overnight: phospho-Histone H3 antibody diluted 1:200 (Millipore 06-570); anti-Caspase-3 diluted 1:100 (BD Biosciences 559565); anti-Histone H2A.X diluted 1:200 (GeneTex 127342). The following day anti-mouse or anti-rabbit secondary antibody was diluted 1:500 (Alexa Fluor, Invitrogen) and incubated at 4°C overnight. The embryos were then mounted for imaging. For acridine orange (AO; Sigma A6014) staining on WT and zep mutants, a 50x stock (250 μg/ml) of AO was diluted in 0.003% PTU. At specific time points, the 0.003% PTU was removed and replaced with the AO staining solution at room temperature. The embryos were washed three times with 0.003% PTU and imaged in 0.02% tricaine/2% methylcellulose/E3.