Rabbit anti human γ h2ax

Laid embryos were collected as mentioned above. The stage of embryos was staged as described42 (link) and doubly checked by WT or WT-like control embryos (the siblings) that were collected in the same time windows. Embryos were dechorionated and sorted into different dishes of mibⁿⁿ²⁰⁰² homozygotes and WT siblings at 14 hpf. WT and mibⁿⁿ²⁰⁰² homozygotes that did not exposed to UV were moved to room temperate for 15 min. At the same time, the WT-UV control was placed in laminar flow hood with UV on for 15 min. Afterwards, all dishes were moved to 28.5 °C incubator for 1 hour before collected. Collected embryos were dehydrated by −20 °C methanol/acetone (1:1) and stored at −20 °C. Embryos were then sorted into 10 embryos/vial and washed by 1% Triton/PBS several times on shaker. After overnight blocking, embryos were washed by 2% BSA/1% Triton/PBS at 4 °C on shaker, and 1:1000 rabbit anti-human γ-H2AX (Cell signaling, #9718) in 2% BSA/1% Triton/PBS was used for overnight reaction. After intensive wash, 1:1000 goat anti-rabbit Alexa-488 antibody (Molecular probe) in 2% BSA/1% Triton/PBS was used for overnight reaction. After intensive wash, embryos were fixed by 4% paraformaldehyde. Embryos were further deyolked and flat-mounted in 75% glycerol to avoid strong background fluorescence from yolk. Images were taken by Zeiss Axiovision Imager A1.