Purification of Mouse Brain Vessels

The overall goal of the procedure is to purify the vascular compartment of the mouse brain while maintaining its structural integrity, using a series of low speed centrifugations and filtrations without the need for specific antibodies or transgenic strains. Many efforts have been made to develop techniques allowing cellular, molecular, and pharmacological investigations of the blood brain barrier. The absence of separation, gradients, colons, or high speed centrifugation steps makes the purification of brain vessels by this procedure very easy, rapid, and inexpensive.

The brain vessels will remain structurally intact with the endot cells sealed by tag junction and surrounded by the basal lanar parasites, vascular S most muscle cells, and perivascular astro membranes. This protocol can help answer key questions regarding the function of the CE vascular compartment and allowing the study of its molecular composition demonstrating the procedure will be an ole a postdoc from the Martin Coen S team. Before beginning the procedure, cut the bottom off of the upper screwing part of the filter holder.

Then use a scalpel to inci the skin from the neck of the mouse to the nose and pull the skin back. Wash the skull with PBS to remove any contaminating hairs, and then insert a pair of scissors into the skull anterior to the olfactory bulbs. Open the scissors to rupture the skull into parts and remove the brain with the spatula.

Next, dissect out the choroid plexus from the lateral ventricles and transfer the brain into a 150 milliliter beaker containing 20 milliliters of B one solution on ice. Then use two scalpels to vigorously chop the brain into two millimeter fragments of tissue, and use an automated down homogenizer to homogenize the tissue for 20 strokes at 400 RPM on ice. To purify the vessels, transfer the homogenate into a 50 milliliter plastic tube and spin down the tissue slurry.

In a swinging rotor centrifuge, a large white interface consisting mostly of myelin will form on the top of the vessel pellet. Discard the supernatant at 20 milliliters of ice cold B two solution, and shake the tube vigorously for one minute. After a second centrifugation, the myelin will form a dense white layer at the surface of the supernatant.

Slowly rotate the tube to allow the B two solution to run along the wall as it is poured out and discard the myelin with the supernatant. Then use a plastic pipette wrapped in absorbent paper to remove any residual fluid from the walls of the tube. Taking care not to touch the vessel pellet and turn the tube upside down on ice.

Next, resus, suspend the pellet in one milliliter of ice cold B three solution, followed by the addition of another five milliliters of B three solution taking care that the vessels do not form aggregates. Now place a 20 micron nylon mesh filter on top of a Becker flask and equilibrate the filter with 10 milliliters of ice cold B three solution. Pour the vessel preparation onto the filter and carefully rinse the vessels with another 40 milliliters of ice cold B three solution.

Then use clean forceps to immediately transfer the filter into a beaker containing 30 milliliters of fresh B three solution, and attach the vessels from the filter with gentle shaking. Pour the beaker contents into a 50 milliliter plastic tube. Then after a centrifugation, re suspend the pellet in one milliliter of ice cold B three solution before immuno staining.

Transfer the vessels into 0.2 milliliter PCR tubes and place the tubes on ice until the tissue sediment in the bottom of the tubes. Then use gel loading tips to aspirate most of the liquid. Next, insert a Siliconized 40 micrometer glass capillary into a P 200 pipette tip, and use the capillary to transfer the vessels to a glass slide when the vessels have settled carefully remove the liquid with a piece of absorbent paper.

Then load a single drop of mounting medium onto the vessels and apply a cover slip at a 45 degree angle, allowing the mounting medium to spread along the edge of the glass until the cover slip is in place. In this image, continuous RIN labeling around neural endothelial cells can be observed confirming the retention of the basal lamina on the purified vessels. Components of the endothelial tight junctions, such as ZO one, are detected on the purified vessels as well.

Further, NG two and smooth muscle actin labeling indicate the retention of intact parasites and vascular smooth muscle cells respectively. Perivascular astro glial membrane proteins connection 43 and Aquaporin four are also detected at the surface of the purified vessels, demonstrating the additional retention of perivascular astrocyte membranes during the purification process, only dispersed and short GFAP positive fibers, however, are observed on the isolated vessels revealing the astrocyte cell bodies are not co purified. Only few neuronal fibers remain attached to the vessels.

Similarly, IBA one and oli two positive cells are also not detected confirming that microglia and oligodendrocytes are not co purified. We strongly recommend removing the current plexus before homogenization of the brain as we experienced it as a possible source of contamination, work in a clean area, and avoid hair and dust contamination. As antibodies have a high nonspecific affinity for these materials.

It is also important to always perform a nuclear staining as well. It is possible to remove blood cells from the purified vessels by performing intracardiac perfusion with PBS prior to the vessel extraction. We have successfully extracted airs and proteins from vessel purified by this procedure from both freshly prepared or frozen vessel palettes.

We have also used fluorescent probes and confocal microscopy to study the ex vivo endothelial transport function of the vessels. We wish you good luck with your experiments.