Cells were fixed as described in [24 (link)] with some modifications. Briefly, cells were collected in centrifuge tubes and fixed for 30 minutes in Solution I (10 mM EGTA, 25 mM HEPES, 2 mM MgCl2, 60 mM PIPES pH 6.9 (PHEM 1X); formaldehyde 1% (Sigma-Aldrich), Triton X-100 2.5%, Sucrose 4%), and for 10 minutes in solution II (PHEM 1X, formaldehyde 4%, Triton X-100 1.2%, Sucrose 4%). Following blocking in 3% bovine serum albumin (Sigma-Aldrich) supplemented Tris buffered saline (10 mM Tris pH 7.4, 0.15 M NaCl) -Tween 20 0.1% (TBST) for 10 minutes, fixed cells were incubated overnight at room temperature with primary antibodies: rabbit anti-H3K9me3 (07–442, Millipore; 1:200), rabbit anti-H3K27me3 (07–449, Millipore; 1:500). After two washes in TBST 3% BSA, cells were labeled with Alexa Fluor 568-conjugated goat anti-rabbit IgG (Invitrogen, catalog number #A-11036, 1:500) for 1 h, stained with 1 μg/mL Hoechst for 5–10 minutes, washed in TBST and finally mounted in Citifluor AF2 glycerol solution (Citifluor Ltd, London).
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