Immunostaining for oligodendrocyte-specific galactosphingolipids in fixed brain sections using the cholesterol-selective detergent digitonin.
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.
Matsubayashi
Yutaka
Y
Department of Molecular and Systems Neurobiology, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, Japan.
Iwai
Lena
L
Toda
Tomohisa
T
Lu
Qing Richard
QR
Kawasaki
Hiroshi
H
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
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