Aortic Root Lesion Quantification

At the study endpoint, hearts were removed from each mouse following PBS perfusion. After a brief PBS wash, individual mouse hearts were embedded in OCT and stored at −80°C until further use. About 8–10-micron serial sections of the aortic root were obtained by cutting the OCT-embedded hearts, as described earlier (27 (link)). Care was taken to ensure that only serial sections collected from aortic root regions representing about 100–150 microns following the valve leaflet were utilized in all morphometric experiments. Additional care was exercised to ensure that staining was performed on sections within similar regions of the aortic root across all treatment groups for quantification and comparison. Aortic root sections were concurrently stained with 0.5% w/v Oil red O (ORO) and hematoxylin and eosin (H&E) to assess the lipid burden and plaque area, respectively. Additional sections were utilized for the detection of collagen content within aortic root lesions using Masson’s Trichrome stain kit (Polysciences). For ORO-stained and MT-stained sections, hematoxylin counterstaining was utilized. All sections were mounted with DPX mounting media, observed using Olympus BX61VS microscope and images were captured using 10× magnification. For quantitative morphometry, eight animals per genotype for each sex with at least 30 sections within each group were analyzed.