Sudan 4

The extent of atherosclerosis was expressed as the percentage of plaque area in the aorta, which was determined by en face quantification as described in the literature.^{[16 (link)]} Briefly, after fixation with 10% neutral formalin, the aorta was cut longitudinally, unfolded, and rinsed in 70% ethanol (the volume of absolute ethyl alcohol [Sigma-Aldrich]: the volume of distilled water = 7:3) for 3 min. Then, the aorta was stained with Sudan IV solution, which consisted of 0.1% Sudan IV (AMRESCO, Solon, OH, USA), 35% ethanol, and 50% acetone (Sigma-Aldrich) for 3 to 5 min, followed by destaining in 80% ethanol for 3 to 5 min. Thereafter, the stained aorta was splayed and pinned flat on a white plate. Images were captured using a digital camera (Canon Inc, Tokyo, Japan) in a fixed parameter and transferred to the computer. The atherosclerotic lesion area stained by Sudan IV (red stain) and total aortic surface area were measured using a computer-assisted image analysis system (NIH, Bethesda, Maryland, USA).