Rat anti mouse cd144

Eight to 10 weeks old male mice were euthanized and pressure perfused with saline through the left ventricle followed by perfusion with 5 ml of chilled 4% paraformaldehyde [23 (link)] solution. The aortic tree was carefully dissected and opened longitudinally to expose the lumen. The tree was then sandwiched between two glass slides and fixed in 4% PFA for another 2 h on ice followed by an overnight blocking with PBS containing 1% BSA. All primary antibodies were prepared in 4 ml of PBS containing 1% BSA and 5% normal serum. The following primary antibodies were used, Rat anti Mouse CD144 (555,289, BD Biosciences), and Rabbit Monoclonal anti-YAP (14,074, Cell Signaling). Samples were washed with PBS/Tween20 for 2 h (12x10min washes). All secondary antibody were used at 1:2000 dilution and incubated for 2 h at room temperature and washed for 2 h before staining with DAPI for 10 min. Aorta with lumen facing up were mounted and cover slipped using Prolong Gold. Images were captured at 63X using Confocal microscope (Leica TCS SP5) using a HCX PL APO Lbd Bl 63x/1.40–0.60 NA objective. All images for p65, eNOS, ICAM and ARHGAP18 were captured at the endothelial cell layer as identified by VE-Cadherin staining.