αh3k27ac

Dechorionated embryos were transferred into a 1.5 ml tube with 500 μl heptane, and then 500 μl of freshly prepared 4% formaldehyde in PBS was added. The tube was vigorously shaken for 20 min at room temperature. After removing the lower aqueous layer carefully and as much as possible, 500 μl methanol was added, and the mixture was shaken for 1–2 min. Devitellinized embryos which sank to the bottom were kept and washed with methanol for three times. Fixed embryos were stored at −20 °C in methanol until use. To perform immunostaining, fixed embryos were rehydrated with 500 μl PBST (0.3% Tween-20) for 5 min at room temperature four times. Embryos were blocked in PBST with 3% donkey serum (Sigma-Aldrich) for 30 min. 1:500 rabbit α-H3K27ac (Active Motif, #39133) was then added and incubated at 4 °C overnight. Next, embryos were washed with PBST for 15 min three times, incubated with 1:500 anti-rabbit Alexa Fluor 488 (Thermo Fisher, #A11008) at room temperature for 1 h, washed with PBST for 15 min three times, and mounted on a glass slide in Fluoromount (Sigma-Aldrich, #F4680). Hoechst 33258 (Thermo Fisher, #H3569) was added at 1:2000 during the second wash after secondary antibody incubation.