Isolating Mouse Brain Vessels: A Protocol to Isolate Intact Vessels from Murine Brain Sample
Transcrição
Brain vessels are surrounded by a layer of tightly connected endothelial cells. These cells have a basement membrane around them along with cellular components such as pericytes and astrocytes.
To isolate brain vessels from surrounding neurons and glial cells, take a beaker containing mouse brain submerged in a homogenization buffer. Mince it into small pieces. Homogenize the brain tissue to dissociate it, releasing neurons, glial cells, and brain vessels. This treatment also disrupts myelin sheath surrounding the neurons.
Transfer the homogenate to a tube and centrifuge to pelletize the vessels. The lighter components such as neurons and glial cells remain in the supernatant while myelin forms a dense interface layer surrounding the pellet.
Discard the supernatant and resuspend the vessel pellet with the attached myelin layer in a suitable density gradient medium. Centrifuge to pellet the vessels and obtain a band of fat-rich myelin floating at the top of the supernatant. Remove the myelin band along with spent media and resuspend the pellet in a fresh buffer.
Strain the crude vessel preparation using a filter assembly containing a filter small enough that the vessels cannot pass through it. Transfer the filter into a fresh beaker containing buffer solution. Gently scrape the filter paper to detach the vessels and store them for further analysis.
