Vectorshield mounting medium for fluorescence

For tissue processing, aortas were fixed overnight in 3% PFA at 4°C. Fixed aortas were placed in a solution of 30% sucrose in PBS and stored at 4°C overnight. Aortas were then gently agitated in embedding media (1:2 ratio of 30% sucrose in PBS in optimal cutting temperature (OCT) medium and then frozen in the same media using 2-methylbutane and liquid nitrogen. Samples were then cut into 8 μm sections using a Cryostat, mounted onto glass slides and coverslips attached using Vectorshield mounting medium for fluorescence (Vector Laboratories, Burlingame, CA). For cholesterol visualization, samples were stained with 50 μg/mL of filipin in PBS for 1 hr at room temperature. For F4/80 labeling, sections were stained with primary antibody at 1:500 dilution overnight at 4°C, followed by A546-anti-rat secondary antibody at 1:500 dilution for 4 hours at room temperature. For adoptive transfer of biotin-fluorescein-dextran labeled BMMs to Apoe^−/− mice, tissues were stained with 1 mg/mL of streptavidin-A546 for 30 min and nuclei stained using 1 μg/mL of Hoechst for 30 min.