Assessment of Microvessel Isolation Protocol by Follow-on Immunostaining and Confocal Microscopy to Visually Gauge the Level of Capillary Enrichment and the Inclusion of Vessels containing alpha-Smooth Muscle Actin-positive Cells.

Validation study of murine brain microvessel collection methods comparing the output from samples generated from: Group 1--whole brain, Group 2--enrichment by density gradient centrifugation (DSG), and Group 3--DSG plus follow-on filtration. Microvessel fragment output from each protocol was analyzed by applying the following protocol: fixation by 4% paraformaldehyde in PBS, immunostaining for specific vascular markers (Endothelial cells--IsolectinB4 or PECAM-1/CD31, Mural cells-- PDGFRbeta or alpha Smooth Muscle Actin/aSMA). A subset of samples were collected from NG2:DsRed/+ animals. Cell nuclei were labeled with DAPI.