Protocol for isolation of the brain vasculature from the mouse cortex for proteomics

The neurovascular unit, composed of brain endothelial cells, pericytes, and astrocytes, regulates molecular exchange between blood and the central nervous system. Here, we present a protocol to isolate brain vessels from the mouse cortex using mechanical homogenization and dextran density-gradient centrifugation. We describe steps for cardiac perfusion, meningeal and white matter removal, homogenization, and separation of capillary-depleted parenchyma. We detail procedures for validating purity by immunofluorescence and western blot followed by sample preparation for liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics. For complete details on the use and execution of this protocol, please refer to Huang et al. 1