Isolation and Imaging of Microvessels From Brain Tissue.

Proper brain function depends on the integrity of the blood-brain barrier (BBB), which is formed by a specialized network of microvessels in the brain. Reliable isolation of these microvessels is crucial for studying BBB composition and function in both health and disease. Here, we describe a protocol for the mechanical dissociation and density-based separation of microvessels from fresh or frozen human and murine brain tissue. The isolated microvessels retain their molecular integrity and are compatible with downstream applications, including fluorescence imaging and biochemical analyses. This method enables direct comparisons across species and disease states using the same workflow, facilitating translational research on BBB biology. Key features • The protocol employs mechanical dissociation and density-based separation to isolate microvessels from brain tissues. • The protocol was used to study molecular changes in brain microvessels in neurodegeneration and aging. • Validated downstream applications of this method include fluorescence imaging, RNA sequencing, proteomics, western blotting, and ELISA. • The protocol can be applied to fresh and frozen human and murine brain samples.