4.4. En Face Lesion Analysis

The extent of atherosclerosis development was quantified by en face lesions analysis according to described procedures [33]. After sacrificing mice, we separated the aorta and fixed in 4% paraformaldehyde overnight at 4 °C. After the aorta was washed for 3 min by using 70% ethanol, atherosclerotic plaque was stained for 10 min by using 0.5% Sudan IV (S100286, Aladdin, Shanghai, China) and washed twice for 3 min by using 80% ethanol. The aorta was washed with PBS (pH 7.4). Finally, the adventitial fat was removed and the aorta was opened longitudinally. Digital images were monitored by stereoscopic microscopes and analyzed with the ImageJ (1.8.0) software. The extent of lesions in the entire aorta was calculated, expressed as a percentage of the total aortic surface area covered by lesions.