Isolation of Mouse Brain Microvessels

Mouse brain microvessels were isolated as previously described (Park et al., 2013 (link); Andras et al., 2017 (link)). Briefly, brains were homogenized in cold isolation buffer (102 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 15 mM HEPES, 25 mM NaHCO₃, 10 mM glucose, 1 mM Na pyruvate) with freshly added protease inhibitors (Thermo Fisher Scientific) and filtered through a 300 μm nitrocellulose mesh filter (EMDMilipore). Then, 26% dextran (MW, 150 kDa) in isolation buffer was added to the filtered brain homogenate, mixed, and centrifuged at 5,800 × g for 20 min at 4°C. The supernatant was removed, pellets were resuspended in isolation buffer, and filtered through a 120 μm nitrocellulose mesh filter (EMDMilipore). Filtered homogenates were then re-pelleted by centrifugation (1,500 × g, 10 min, 4°C) and re-resuspended either in 200 μL of radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) supplemented with protease inhibitors for immunoblotting, or in PBS for RNA sequencing.