A Method for the Isolation of Neurovascular Units from Rat Brain
Trascrizione
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 tissue using 15 up-and-down strokes at 3,700 RPM. Then, pour the homogenate into labeled centrifuge tubes. Between samples, use 70% ethanol to clean the pestle.
To carry out centrifugation, add 8.0 milliliters of 26% dextran solution to each labeled centrifuge tube containing brain homogenate. Invert the tube twice and then thoroughly vortex the sample. Conduct the vortexing of each sample using multiple angles to ensure a thorough mixing of brain homogenate solution with 26% dextran solution. Centrifuge the samples at 5,000 g and 4 degrees Celsius for 15 minutes.
Aspirate the supernatant and resuspend the pellet in 5.0 milliliters of BMB with protease inhibitor cocktail. Then, vortex the pellet to ensure thorough mixing. Next, add 8.0 milliliters of 26% dextran to each centrifuge tube and vortex as just demonstrated. Then, centrifuge the samples again.
Using a vacuum flask and glass pipette, aspirate the supernatant and ensure that the pellet containing the brain microvessel is not disrupted. Resuspend the pellet in BMB with protease inhibitor and then 26% dextran two more times. After the final centrifugation, add 5.0 milliliters of BMB to each pellet and vortex to resuspend the sample.
