A Method for the Isolation of Neurovascular Units from Rat Brain

Begin with a cold glass mortar containing rat cortical brain tissue, which is the brain's outermost layer.

Add a brain microvessel buffer or BMB, containing protease inhibitors.

Now, move the pestle up and down to dissociate the tissue.

This agitation releases the neurovascular units, or NVUs, comprising microvessels, pericytes, neurons, and glial cells. The protease inhibitors block the released proteases, protecting the microvessels.

Transfer this mixture to a tube.

Add a dense polysaccharide solution. Use a vortex to mix the solution with other contents uniformly.

Centrifuge. The dense polysaccharides allow sedimentation of NVUs, which are denser than tissue debris.

Discard the supernatant and resuspend the NVUs in BMB containing protease inhibitor and polysaccharide solution.

Use the vortex to mix the contents.

Repeat the centrifugation process. Discard the supernatant to remove any residual debris.

Resuspend the NVUs in BMB and store them for further analysis.

Place the cortical brain tissue into a chilled glass mortar. Then, add 5 milliliters of BMB with protease inhibitor cocktail. Using an overhead power homogenizer, insert a pestle into the mortar and homogenize the brain