Macrophage Proliferation and Apoptosis Quantification

To quantitate macrophage proliferation and apoptosis, immunofluorescence staining was performed using a standard protocol^{30 (link)}. Briefly, 10 micrometer (μm) thick frozen aortic sections were brought to the room temperature (RT), fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% normal goat serum and 3% bovine serum albumin. Subsequently, sections were incubated with anti-Ki-67 (1:750 dilution, abcam, Cambridge, MA) or activated caspase-3 (1:1000 dilution, abcam) together with anti-Mac-3 (1:400 dilution, BD Biosciences, San Jose, CA) overnight at 4°C. After washing with phosphate buffer saline (PBS), corresponding secondary antibodies (goat anti-rabbit (Alexa Fluor® 488, Life Technologies, Waltham, MA) and goat anti-rat (Alexa Fluor® 594, Life Technologies) were applied for 1 hour at room temperature and cover slips were mounted with fluoroshield containing DAPI (Sigma, St. Louis, MO). The images were taken using Nikon (Eclipse Ti-s) and Zeiss LSM 900 confocal microscopes.