Oil Red O staining of whole aortas for en face analysis
For atherosclerotic lesion analysis, nine mice per group were evaluated. The aortas from the base of the ascending aorta to the iliac bifurcation were separated, and the aortic roots with the heart were harvested. For en face aorta analyses, the aortic tree was perfused with PBS, and the aorta was then dissected en bloc from the root to the iliac bifurcation by removing minor branching arteries and fat tissue. The aortic lumen was opened with a longitudinal incision and then immediately fixed in 4% paraformaldehyde. After 24 h of fixation, aortic lipids were stained with Oil Red O (Solarbio, Cat#G1261), and the stained aortas were photographed. The percentage of aortic area stained with Oil Red O was determined and quantified with Image‐Pro Plus 6.0 (Image Metrology, Copenhagen, Denmark, RRID: SCR_007369).
Atherosclerotic lesion analysis andimmunofluorescence staining of aortic sinus
The heart and proximal aorta were collected and embedded in optimum cutting temperature compound. Sections were stained with Oil Red O and hematoxylin for analysis of plaque sizes. Masson Trichrome staining was used to analysis collagen content. For immunofluorescence staining, slides were fixed in cold methanol and permeabilized using 0.3% Triton-X. Then, slides were blocked using 5% bovine serum albumin for 30 min and incubated overnight with primary antibodies. Alexa-488/647 Alexa647(Abcam, Cat#ab150075, RRID:AB_2752244), Alexa488(Abcam, Cat#ab150077, RRID:AB_2630356) conjugated secondary antibodies were used for detection. The isotype antibodies were used as controls. Slides were counterstained with DAPI(Beyotime, Cat#P0012, China).