title: Immunostaining for oligodendrocyte-specific galactosphingolipids in fixed brain sections using the cholesterol-selective detergent digitonin.
abstract: Galactocerebroside (GalC) and its sulfated derivative sulfatide (SUL) are galactosphingolipids abundantly expressed in oligodendrocytes (OLs). Despite their biological importance in OL development and function, attempts to visualize GalC/SUL in tissue sections have met with limited success. This is at least in part because permeabilization of tissue sections with detergents such as Triton X-100 results in significant degradation of GalC/SUL immunoreactivity. Here we establish a novel method that enables visualization of endogenous GalC/SUL in OLs and myelin throughout the entire depth of brain sections. We show that treating brain sections with the cholesterol-specific detergent digitonin instead of Triton X-100 or methanol leads to efficient antibody penetration into tissue sections without disrupting GalC/SUL immunoreactivity. We also determine the optimal concentrations of digitonin using confocal microscopy. With our method, the morphology and the number of GalC/SUL-expressing OLs can be visualized three-dimensionally. Furthermore, our method is applicable to double immunostaining with anti-GalC/SUL antibody and other antibodies which recognize intracellular antigens. Our simple method using digitonin should prove to be useful in enabling detailed examination of GalC/SUL expression in the brain in both physiological and pathological conditions.
authors.affiliation: Department of Molecular and Systems Neurobiology, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Matsubayashi
authors.forename: Lena
authors.initials: L
authors.lastname: Iwai
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Toda
authors.forename: Qing Richard
authors.initials: QR
authors.lastname: Lu
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawasaki
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D000071183:Autophagy-Related Proteins; D001921:Brain / Q000166:cytology*; D003902:Detergents / Q000494:pharmacology*; D004072:Digitonin / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005699:Galactosylceramides / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D004676:Myelin Basic Protein / Q000378:metabolism; D019081:O Antigens / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D009836:Oligodendroglia / Q000187:drug effects* / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D013433:Sulfoglycosphingolipids / Q000378:metabolism
chemical_list: D000071183:Autophagy-Related Proteins; D003902:Detergents; D005699:Galactosylceramides; D047908:Intracellular Signaling Peptides and Proteins; D004676:Myelin Basic Protein; D019081:O Antigens; C428743:Rb1cc1 protein, mouse; D013433:Sulfoglycosphingolipids; C002454:galactocerebroside; D017830:Octoxynol; D010751:Phosphopyruvate Hydratase; D004072:Digitonin