Rabbit anti β catenin antibody

Prior to excision, aortae were perfused serially via the left ventricle of the heart with: i) 0.5 mM EDTA in PBS; ii) 4% paraformaldehyde, 7.5% sucrose, 0.5 mM EDTA, in PBS; and iii) 0.5 mM EDTA in PBS, respectively. Dissected aortae were fixed in 4% paraformaldehyde for 30 minutes and then permeablized with 0.1% Triton X-100 in PBS at room temperature, followed by an overnight incubation (4°C) with a rabbit anti-β-Catenin antibody (Sigma #C2206) in 1:1000 diluted blocking solution (Dako-Agilent, K800621, Carpinteria, CA). Secondary anti-rabbit IgG antibody conjugated with Alexa Plus 555 (Thermo #14–387-071, Waltham, MA) at 1:1500 dilution was used for visualization of endothelial borders. Nuclei were counter stained with 1μg/mL Hoechst 33342 (Cell Signaling #4082). Vessels were mounted on a coverslip with ProLong anti-fade mounting medium (Thermo #P36962, Waltham, MA). Images were acquired with confocal microscope (Carl Zeiss) and ZEN 2012 software (Carl Zeiss). Since our cell culture work revealed no impact of sex, composite data from en face staining was typically sampled 4–8 times in aortae isolated from 4–5 mice of either sex. Representative staining in Figure 1 was derived from a male mouse and that in Figure 5 representes a female mouse.