Development of rapid staining protocols for laser-capture microdissection of brain vessels from human and rat coupled to gene expression analyses.
Laser-capture microdissection (LCM) is a technique that enables selective extraction of desired cells from heterogeneous tissues compatible with subsequent molecular analyses. The specific visualization of desired cell types prior to LCM is essential for achieving selective capture. We have developed rapid and selective staining protocols for LCM extraction of microvessels from human and rat brain. Vessels in human and rat brain sections were visualized by a 2 min exposure to fluorescein-labeled lectins Ulex Europeaus Agglutinin I (UEA I) and Ricinus Communis Agglutinin I (RCA I), respectively. Immunohistochemical staining for the endothelial-specific marker, Factor VIII-related antigen (FVIII-rAg), co-localized with that for either UEA I or RCA I, confirming the selective staining of vascular structures with these lectins. Both brain vessels and perivascular parenchyma were captured using LCM, followed by RNA isolation. RT-PCR analyses demonstrated the enrichment of LCM-captured vessels and parenchyma in FVIII-rAg and GFAP mRNA, respectively. LCM-captured human vessels also expressed the tight junction-specific gene, zonula occludens 1 (ZO-1). LCM extraction of vessels from brain sections can be used to perform molecular fingerprinting of neurovascular unit in various brain pathologies.
Mojsilovic-Petrovic
Jelena
J
Cerebrovascular Research Group, Institute for Biological Sciences, National Research Council of Canada, 1200 Montreal Road, Building M-54, Ottawa, Ont., Canada K1A 0R6.
Nesic
Momir
M
Pen
Ally
A
Zhang
Wandong
W
Stanimirovic
Danica
D
D000818:Animals; D001808:Blood Vessels / Q000378:metabolism*; D001921:Brain / Q000166:cytology* / Q000378:metabolism; D005260:Female; D015870:Gene Expression / Q000502:physiology*; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D042282:Microdissection / Q000379:methods*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D037121:Plant Lectins / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013194:Staining and Labeling / Q000379:methods*; D062826:Zonula Occludens-1 Protein; D014841:von Willebrand Factor / Q000378:metabolism
D005904:Glial Fibrillary Acidic Protein; D008565:Membrane Proteins; D010750:Phosphoproteins; D037121:Plant Lectins; D012333:RNA, Messenger; C017707:Ricinus communis agglutinin-1; C569042:TJP1 protein, human; C569044:Tjp1 protein, rat; C036850:Ulex europaeus lectins; D062826:Zonula Occludens-1 Protein; D014841:von Willebrand Factor