Immunostaining for oligodendrocyte-specific galactosphingolipids in fixed brain sections using the cholesterol-selective detergent digitonin.
Authors: Matsubayashi, Y., Iwai, L., Toda, T., Lu, Q.R. and Kawasaki, H.
Journal: J Neurosci Methods
Volume: 178
Issue: 1
Pages: 87-98
ISSN: 0165-0270
DOI: 10.1016/j.jneumeth.2008.11.018
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.
Source: PubMed
Immunostaining for oligodendrocyte-specific galactosphingolipids in fixed brain sections using the cholesterol-selective detergent digitonin
Authors: Matsubayashi, Y., Iwai, L., Toda, T., Lu, Q.R. and Kawasaki, H.
Journal: JOURNAL OF NEUROSCIENCE METHODS
Volume: 178
Issue: 1
Pages: 87-98
eISSN: 1872-678X
ISSN: 0165-0270
DOI: 10.1016/j.jneumeth.2008.11.018
Source: Web of Science (Lite)
Immunostaining for oligodendrocyte-specific galactosphingolipids in fixed brain sections using the cholesterol-selective detergent digitonin.
Authors: Matsubayashi, Y., Iwai, L., Toda, T., Lu, Q.R. and Kawasaki, H.
Journal: Journal of neuroscience methods
Volume: 178
Issue: 1
Pages: 87-98
eISSN: 1872-678X
ISSN: 0165-0270
DOI: 10.1016/j.jneumeth.2008.11.018
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.
Source: Europe PubMed Central