Ma1 065

Cell-agarose-collagen scaffolds were fixed overnight in Histochoice tissue fixative (VWRVH102, VWR). Samples were cryo-embedded using OCT compound and cryo-sectioned at 20 μm (CM1950, Leica). Slides were immunolabelled, following the immunohistochemistry protocol previously described, with primary antibodies raised against the NG2/CSPG4 intracellular domain (1:200, GGQPDPELLQFCRTPNPALRNGQYWV, UIC Protein Core), NG2/CSPG4 ectodomain (1:200, AB5320, Sigma-Millipore), MMP-13 (1:200, AB39012, Abcam) and/or CHC (1:200, MA1-065, Thermo Fisher). Images were acquired using a laser scanning confocal microscope with 63 × oil immersion objective (LSM 710, Zeiss, White Plains, NY, USA). The laser intensity, brightness, and gain were standardised for all image acquisitions. Colocalisation coefficients and fluorescence/channel intensity were calculated using ImageJ (Rasband, 2011 ) using the Pearson’s overlap coefficient were compare (Adler and Parmryd, 2010 (link)) and the Costes method. Images were quantified by personnel blind to the study design, outcome assessment, and analysis. Four biological replicates and 5 technical replicates for each biological replicate was used for each experimental group.

Brain microvessels were isolated from young (3-month-old) and aged (22-month-old) mice as previously described^{11 (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.