期刊名称:Proceedings of the National Academy of Sciences
印刷版ISSN:0027-8424
电子版ISSN:1091-6490
出版年度:1977
卷号:74
期号:9
页码:3782-3786
DOI:10.1073/pnas.74.9.3782
语种:English
出版社:The National Academy of Sciences of the United States of America
摘要:An immunoelectron microscopic method is described for sensitive high-resolution visualization of tissuebound cholera toxin. The principle is to incubate cells or tissue sections with toxin and then to localize the bound toxin with toxin-specific peroxidase (donor:hydrogen-peroxide oxidoreductase; EC 1.11.1.7 )-conjugated antibody and enzyme substrate. Thin sections are examined for electron-opaque precipitates in a transmission electron microscope. Because of the specific binding of the toxin to membrane ganglioside GM1, the method can be used for ultrastructural localization of this ganglioside. Semiquantitative data are obtained by titration of the limiting concentration of cholera toxin producing specific precipitates. The specificity of the method was controlled in various ways, including analyses of the correlation between the immunoelectron microscopy results and determinations of ganglioside GM1 in tissues with different ganglioside concentrations, tissues hydrolyzed with Vibrio cholerae sialidase, tissues in which exogenous GM1 has been incorporated, and lipid-extracted tissues. The immunoelectron microscopic method demonstrates that membrane GM1 ganglioside is positioned on the external side exclusively. Cell-bound toxin remains in its original location on the plasma membrane surface of cells below 18{degrees