TY - JOUR
T1 - Immunostaining for oligodendrocyte-specific galactosphingolipids in fixed brain sections using the cholesterol-selective detergent digitonin
AU - Matsubayashi, Yutaka
AU - Iwai, Lena
AU - Toda, Tomohisa
AU - Lu, Qing Richard
AU - Kawasaki, Hiroshi
PY - 2009/3/30
Y1 - 2009/3/30
N2 - 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.
AB - 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.
KW - Cholesterol-specific detergent
KW - Digitonin
KW - Galactocerebroside
KW - Galactosphingolipid
KW - Immunohistochemistry
KW - Oligodendrocyte
UR - http://www.scopus.com/inward/record.url?scp=59249105383&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=59249105383&partnerID=8YFLogxK
U2 - 10.1016/j.jneumeth.2008.11.018
DO - 10.1016/j.jneumeth.2008.11.018
M3 - Article
C2 - 19100769
AN - SCOPUS:59249105383
VL - 178
SP - 87
EP - 98
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
SN - 0165-0270
IS - 1
ER -