Immunostaining of H3K27ac and H3K27me3

Immunostaining, image acquisition, and analyses were performed as described previously (Inoue et al, 2018 (link)). Embryos were fixed with 3.7% paraformaldehyde and 0.2% Triton X‐100 for 20 min at room temperature. After washing 4 times with PBS containing 1% BSA, samples were incubated with the rabbit anti‐H3K27ac antibody (1/500; Millipore, 07–360) and/or the mouse anti‐H3K27me3 antibody (1/500; Active Motif, 61017) as primary antibodies at 4°C overnight. Then, samples were washed with PBS/BSA and incubated with Alexa flour 568 donkey anti‐rabbit IgG and/or Alexa flour 488 donkey anti‐mouse IgG (1/200; Life Technologies) as secondary antibodies for 1 h at room temperature. After washing with PBS/BSA, samples were mounted on a slide containing VectaShield Antifade Mounting Medium with DAPI (Vector Laboratories). Fluorescent intensities were quantified with Zeiss Zen software.