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).