Selective capture of endothelial and perivascular cells from brain microvessels using laser capture microdissection.
Laser capture microdissection (LCM) of the major cell types comprising brain microvessels offers a powerful technology to explore the molecular basis of the blood-brain barrier in health and disease. However, the ability to selectively retrieve endothelial or perivascular cells, without cross-contamination from the other, has proven difficult. Additionally, histochemical methods previously described for use with LCM have not allowed for identification of all the different size branches of the microvascular tree. Here, we describe a double immunostaining method, combining bright-field and fluorescence microscopy, and using an extensive dehydration with xylene, to clearly identify and spatially resolve endothelial from perivascular cells within all size microvascular branches in frozen brain sections. LCM of these sections, coupled with RNA analysis by reverse-transcription polymerase chain reaction, revealed that captured endothelial cells show endothelial markers but no detectable markers for astrocytes or smooth muscle cells/pericytes. Conversely, captured astrocytes or smooth muscle cells/pericytes demonstrate their respective markers, but not those of endothelial cells. This approach has applicability to microarray analysis, thereby enabling global gene profiling of the different cell types along the entirety of the brain microvascular tree.
Kinnecom
Katie
K
Blood-Brain Barrier Laboratory, Dept. of Pharmacology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA.
Pachter
Joel S
JS
D000199:Actins / Q000096:biosynthesis / Q000378:metabolism; D000818:Animals; D001808:Blood Vessels / Q000166:cytology* / Q000502:physiology; D002196:Capillaries / Q000166:cytology / Q000502:physiology; D002560:Cerebrovascular Circulation / Q000502:physiology*; D004396:Coloring Agents; D004210:Dissection / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D042783:Endothelial Cells / Q000502:physiology*; D005260:Female; D005615:Freezing; D015870:Gene Expression; D007150:Immunohistochemistry; D007834:Lasers; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D012313:RNA / Q000032:analysis / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000199:Actins; D004396:Coloring Agents; D012313:RNA