Preparing Cerebral Endothelial Tubes from Mouse Parenchymal Arterioles

Secure a mouse brain, ventral side up, in a silicon polymer coated-dish containing buffer.

Dissect a tissue section from the hemisphere around the middle cerebral artery, or MCA, with the upper tissue segment extending beyond the branching point from the Circle of Willis.

Secure the tissue with the MCA facing up.

Remove the pia mater, with the arterioles, which branch from the MCA.

Secure the pia mater with the arterioles. Dissect the arteriole and remove the surrounding tissue. Discard any distal branches.

Incubate the arteriole in a proteolytic enzyme cocktail to facilitate partial digestion of the segment.

Replace the enzymes with buffer and transfer the digested segment to a chamber containing buffer on a microscope stage equipped with a trituration pipette.

Pipette the arteriole repeatedly to remove the loosened adventitia, and smooth muscle cells, resulting in an intact tube containing arteriolar endothelial cells.

Place the ventral brain side facing up in a chamber containing cold dissection solution to isolate parenchymal arterioles. To isolate parenchymal arterials, secure the isolated brain with steel pins and cold dissection solution in a Petri dish containing more than