title: Detection of multiple proteins in intracerebral vessels by confocal microscopy.
abstract: Assessment of the blood-brain barrier (BBB) may involve the localization of endothelial proteins within the context of endothelial permeability to plasma proteins. The use of antibodies conjugated to fluorescent dyes, coupled with analysis by confocal microscopy, allows for the detection of multiple proteins in components of the neurovascular unit including endothelium and astrocytes. This chapter provides a detailed protocol for detection of three proteins in fixed or frozen sections of rat brain using three fluorophores with unique excitation/emission spectra. Also included is a protocol for tyramide signal amplification, which is useful for detecting proteins of low abundance, and methods for quantitation of intracerebral vessels expressing a particular protein of interest with and without BBB breakdown to plasma proteins.
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
authors.forename: Janet L
authors.initials: JL
authors.lastname: Manias
authors.forename: Anish
authors.initials: A
authors.lastname: Kapadia
authors.forename: Sukriti
authors.initials: S
authors.lastname: Nag
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001798:Blood Proteins / Q000378:metabolism; D001808:Blood Vessels / Q000737:chemistry / Q000378:metabolism*; D001921:Brain / Q000098:blood supply* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D018613:Microscopy, Confocal; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D051381:Rats
chemical_list: D000906:Antibodies; D001798:Blood Proteins; D005456:Fluorescent Dyes; D011506:Proteins