Quantification of the atherosclerotic lesions

Oil-red O staining was used to quantify the en face atherosclerotic lesion area (6,28). The first segment of the aorta was fixed with 4% paraformaldehyde for 10 min at 37^°C, stained with oil-red O (cat. no. SLBM4444V; Sigma-Aldrich; Merck KGaA) for 1 h at 37˚C, and pinned out flat on a wet black cloth with 0.2-mm diameter stainless steel pins (Fine Science Tools, Inc.). The images of the inner surface of the aorta were captured using a Ziess digital camera (fiber-optic CL 1500 ECO cold light source; Carl Zeiss AG) mounted on a Ziess SteREO Discovery V8 stereo microscope (SteREO Discovery V8; Carl Zeiss AG). The total aortic surface and atherosclerotic lesion areas were analyzed en face using computerized quantitative morphometry by Image Pro Plus software (v6.0, Media Cybernetics, Inc.). The aortic lesion area was expressed as percentage of the total aortic area (29).

To quantify the cross-sectional lesion area, the slices 5-µm from the second segment were stained using EVG (28,30). Images were captured using a Nikon DXM 1200/NIS-Elements mounted on a Nikon Eclipse E600 light microscope (Nikon Corporation) and analyzed using Image Pro Plus software (v6.0, Media Cybernetics, lnc.).