In either confocal or STED mode, immunofluorescence was performed as previously described in [1], using an upright Zeiss microscope with a mounted STEDYCON module and a Zeiss 100x 1.46 NA objective. Primary antibodies used were anti-H3K27ac (Active Motif, 39034) and anti-H3K27me3 (Abcam, ab6002), both at 1:200 dilution. Secondary anti-mouse and anti-rabbit antibodies used were labeled with STAR Red (Sigma-Aldrich, 52283) and STAR Orange (Sigma-Aldrich, 41367), respectively. Excitation was provided by 640 nm (@3%) and 775 nm (@96.5%) lasers for the STAR Red channel, whereas 561 nm (@7.8%) and 775 nm (@100%) lasers were used for the STAR Orange channel. Imaging of the samples was performed with 5 µs pixel dwell time, 64 µm pinhole aperture, 15-line accumulations and a pixel size of 20 nm. Experimental procedures were approved by the EMBL Rome Animal Facility in accordance with European and Italian legislations. 1. Bošković, A., Bender, A., Gall, L., Ziegler-Birling, C., Beaujean, N., & Torres-Padilla, M.-E. Analysis of active chromatin modifications in early mammalian embryos reveals uncoupling of h2a.z acetylation and h3k36 trimethylation from embryonic genome activation. Epigenetics 7, 747–757 (2012).