Rabbit anti h3k27me3

En face staining was performed as previously described in detail 43 (link), with minor modifications 42 (link), 44 (link). Mouse aortas were collected after perfusion with saline and neutrally buffered 4% PFA. Aortic segments were then permeabilized with 0.1% Triton X-100 in PBS for 10 min and blocked with 10% normal goat serum (Invitrogen) in Tris-buffered saline (TBS) containing 2.5% Tween-20 for 30 min at room temperature. Next, aorta segments were incubated with rat anti-VE-Cadherin (1:100; #555289, BD Bioscicence), rabbit anti-EZH2 (#6263, ProSci) or rabbit anti-H3K27me3 (#39155, Active Motif) antibody overnight at 4 °C. After rinsing with washing buffer 3 times, aortic segments were incubated with Alexa Fluor 488-conjugated goat anti-rat and Alexa Fluor 546-conjugated goat anti-rabbit secondary antibodies (1:1,000; Thermo Fisher Scientific) for 1 h in darkness at room temperature. Finally, aortic segments were carefully whole mounted (with lumen side up) in the ProLong Gold-antifade Mounting Media (Invitrogen) for confocal microscopy (IX81, Olympus) with 60x or 40x oil lens. All animal procedures conformed to the Guideline for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of the University of Rochester Medical Center.