Histological and Immunofluorescence Analysis of Zebrafish Embryos

For histological analysis, zebrafish embryos at 48 hpf and 72 hpf were fixed overnight in 4% PFA at 4 °C, dehydrated with a graded ethanol, processed in xylene and embedded in paraffin. Cross section (6 μm) were stained with hematoxylin and eosin using a standard protocol44 (link). For whole mount immunofluorescence staining, zebrafish embryos at 48 hpf were fixed overnight in 4% PFA and dehydrated with methanol. Zebrafish embryos were permeabilized in acetone for 7 min at −20 °C and washed in water, followed by several washes in PBST. After blocking for 30 min in 2% horse serum, zebrafish embryos were incubated with active caspase-3 antibody (BD Biosciences) or p-histone H3 antibody (Millipore) at 4 °C overnight. On the next day zebrafish embryos were incubated with Alexa Fluor 568-conjugated secondary antibody (Invitrogen). Cryosection was performed as described previously45 (link).