Brain microvessels were isolated from young (3-month-old) and aged (22-month-old) mice as previously described11 (link),19 (link),43 (link)–47 (link). To generate sufficient numbers of microvessels, 20 mice of each age were used, with microvessels from four mice pooled in each group (n = 5 groups per age). Microvessels were permeabilized, blocked, stained and mounted as described previously48 . In brief, acutely isolated microvessels were fixed with 4% PFA in PBS at room temperature for 20 min, washed, blocked and permeabilized, and stained overnight at 4 °C with fluorescein-labelled lectin (1:200, Vector Laboratories), mouse anti-clathrin heavy chain Alexa Fluor 647 (X22, 1:100, MA1-065, Thermo Fisher Scientific), and rabbit anti-caveolin-1 (1:200, 3267T, CST) pre-complexed with donkey anti-rabbit Alexa Fluor 555 at a 1:1 ratio. Stained microvessels were subsequently washed with PBS with 1 mM EDTA and mounted with Prolong Gold. Imaging was performed on either a confocal laser scanning microscope (×63 magnification, Zeiss LSM880) or a 3D-structured structured illumination microscope (×100, OMX BLAZE). Microvessels were located at low resolution using only lectin fluorescence; and specific, high-resolution fields of single-plane microvessels were chosen at random for blinded quantification of clathrin and caveolar vesicles normalized to an area of lectin.