To evaluate aortic plaque accumulation, en face analysis was performed on the thoracic portion of aortas using previously described methods [28 (link)]. In brief, formalin-fixed aortas were opened longitudinally and stained with 0.5% oil red O (ORO) in isopropanol for plaque. A blinded observer evaluated an image of the stained aorta, by tracing the total lesion area and the total aortic area, from which a ratio was calculated.
Further analysis of atherosclerosis was evaluated using cryostat sections of the aortic root by previously described methods [28 (link)]. Following heart embedding and sectioning, formalin-fixed sections were stained with 0.5% ORO in propylene glycol and counterstained with hematoxylin and eosin. Images of the ORO-stained aortic roots were analyzed by a blinded observer, in which total lesion area (µm2) was measured. To evaluate the effect of ESM on connective tissue in the aortic root, slides were also stained using a Masson’s trichrome kit (Abcam; Cambridge, MA, USA). A blinded observer measured the total connective tissue area (µm2).
Further analysis of atherosclerosis was evaluated using cryostat sections of the aortic root by previously described methods [28 (link)]. Following heart embedding and sectioning, formalin-fixed sections were stained with 0.5% ORO in propylene glycol and counterstained with hematoxylin and eosin. Images of the ORO-stained aortic roots were analyzed by a blinded observer, in which total lesion area (µm2) was measured. To evaluate the effect of ESM on connective tissue in the aortic root, slides were also stained using a Masson’s trichrome kit (Abcam; Cambridge, MA, USA). A blinded observer measured the total connective tissue area (µm2).