The extracted aortic roots from the mice specimens were processed for histological examination. The specimens were first fixed in 10% neutral buffered formalin solution (Sigma-Aldrich) and subsequently embedded in optimal cutting temperature (OCT) compound (Sakura Finetek). The lipid accumulation within the aortic root sections, cut at 10 µm thickness with a cryostat, was visualized using Oil Red O stain (Sigma-Aldrich). Hematoxylin (Sigma-Aldrich) served as a counterstain to highlight the nuclei.
To assess collagen content, the aortic root sections underwent Masson’s Trichrome and Sirius Red staining protocols, respectively. In Masson’s Trichrome staining, the sections were sequentially treated with Weigert’s iron Hematoxylin (Sigma-Aldrich), Biebrich scarlet-acid fuchsin (Sigma-Aldrich), phosphomolybdic-phosphotungstic acid (Sigma-Aldrich), and aniline blue (Sigma-Aldrich). Sirius Red staining involved an incubation period of one hour in Sirius Red solution (Direct Red 80 in saturated picric acid, Sigma-Aldrich) followed by a wash in acidified water. Post-staining, the sections were dehydrated, cleared, and affixed with a mounting medium (Sigma-Aldrich). The stained sections were subsequently observed under a light microscope and images were captured for further quantitative evaluation.
To assess collagen content, the aortic root sections underwent Masson’s Trichrome and Sirius Red staining protocols, respectively. In Masson’s Trichrome staining, the sections were sequentially treated with Weigert’s iron Hematoxylin (Sigma-Aldrich), Biebrich scarlet-acid fuchsin (Sigma-Aldrich), phosphomolybdic-phosphotungstic acid (Sigma-Aldrich), and aniline blue (Sigma-Aldrich). Sirius Red staining involved an incubation period of one hour in Sirius Red solution (Direct Red 80 in saturated picric acid, Sigma-Aldrich) followed by a wash in acidified water. Post-staining, the sections were dehydrated, cleared, and affixed with a mounting medium (Sigma-Aldrich). The stained sections were subsequently observed under a light microscope and images were captured for further quantitative evaluation.