Immunofluorescence Imaging of H3K27me3

IF was performed as described previously (Tatavosian et al., 2015 (link); Zhen et al., 2014 (link)). Wild-type (PGK12.1), Eed^−/−, Ezh2^−/−, Y-Eed/Eed^−/−, and Y-Ezh2/Ezh2^−/− mES cells were cultured on coverslips and fixed using 2.0% paraformaldehyde. After permeabilizing with 0.2% Triton X-100, the cells were washed with basic blocking buffer (10 mM PBS pH 7.2, 0.1% Triton X-100, and 0.05% Tween 20) and then blocked with blocking buffer (the basic blocking buffer plus 3% goat serum and 3% bovine serum albumin). Anti-H3K27me3 antibody (07–449; Millipore, Billerica, MA) was incubated with the cells for 2 hr at room temperature. After washing with the basic blocking buffer, Alexa Fluor 488-labelled goat anti–rabbit antibody (A-11008; Life Technologies, Carlsbad, CA) was incubated with the cells for 1 hr. After incubating with 0.1 μg/ml hoechst, the cells were washed and then mounted on slides with ProLong Antifade reagents (P7481; Life Technologies, Carlsbad, CA). The images were taken and processed as described previously (Tatavosian et al., 2015 (link); Zhen et al., 2014 (link)).