Alexa fluor 488 anti mouse and 568 anti rabbit iggs

Cells were seeded and grown as described above, fixed in 4% formaldehyde in PBS for 10 min at room temperature, washed three times in PBS, permeabilized for 5 min at room temperature in PBS supplemented with 0.1% Triton X-100 and 0.25% BSA (Sigma-Aldrich), and washed twice in PBS. Corresponding primary antibodies anti-H3K9me3: ab8898 (abcam), anti-H3K27me3: C15410195 (diagenode), anti-GFP: 11814460001 (Millipore) and secondary antibodies (Alexa Fluor 488 anti-mouse and 568 anti-rabbit IgGs from ThermoFisher) were diluted in PBS containing 2% FBS and 0.02% BSA. Primary antibody incubations were performed overnight at 4°C. Secondary antibody incubations were performed for 1h at room temperature. Following antibody incubations, cells were washed once with PBS and incubated for 10 min with PBS containing 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI, 0.5 μg/ml) for 10 min at room temperature to stain DNA. Randomly selected cells were imaged with sequential acquisition settings on a Leica SP5 inverted confocal laser scanning microscope.