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Journal of Histochemistry and Cytochemistry, Vol. 50, 43-56, January 2002, Copyright © 2002, The Histochemical Society, Inc.

ARTICLE

Immunoelectron Microscopic Localization of Cholesterol Using Biotinylated and Non-cytolytic Perfringolysin O

Wiebke Möbiusa, Yoshiko Ohno–Iwashitab, Elly G. van Donselaara, Viola M.J. Oorschota, Yukiko Shimadab, Toyoshi Fujimotoc, Harry F.G. Heijnend, Hans J. Geuzea, and Jan W. Slota
a Department of Cell Biology, University Medical Center Utrecht and Center for Biomedical Genetics, Utrecht, The Netherlands
b Department of Protein Biochemistry, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
c Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, Japan
d Department of Hematology, Division of Thrombosis and Hemostasis, University Medical Center Utrecht, The Netherlands

Correspondence to: Wiebke Möbius, Dept. of Cell Biology, University Center Utrecht and Center for Biogenetics, AZU G02.525, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail: w.moebius@lab.azu.nl

We used a proteolytically modified and biotinylated derivative of the cholesterol-binding {Theta}-toxin (perfringolysin O) to localize cholesterol-rich membranes in cryosections of cultured human lymphoblastoid cells (RN) by electron microscopy. We developed a fixation and immunolabeling procedure to improve the preservation of membranes and minimize the extraction and dislocalization of cholesterol on thin sections. We also labeled the surface of living cells and applied high-pressure freezing and subsequent fixation of cryosections during thawing. Cholesterol labeling was found at the plasma membrane, with strongest labeling on filopodium-like processes. Strong labeling was also associated with internal vesicles of multivesicular bodies (MVBs) and similar vesicles at the cell surface after secretion (exosomes). Tubulovesicular elements in close vicinity of endosomes and the Golgi complex were often positive as well, but the surrounding membrane of MVBs and the Golgi cisternae appeared mostly negative. Treatment of cells with methyl-ß-cyclodextrin completely abolished the labeling for cholesterol. Our results show that the {Theta}-toxin derivative, when used in combination with improved fixation and high-pressure freezing, represents a useful tool for the localization of membrane cholesterol in ultrathin cryosections. (J Histochem Cytochem 50:43–55, 2002)

Key Words: membrane cholesterol, cryosections, electron microscopy, exosomes, cholesterol-enriched microdomains


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