Chapter 15 Immunoelectron Microscopy of Tetrahymena

• Published in: Methods in Cell Biology Vol. 62; pp. 333 - 343
• Main Authors: Gavin, R.H., Hoey, John G., Garcés, Jorge A.
• Format: Book Chapter
• Language: English
• Published: Elsevier Science & Technology 1999
• ISBN: 9780125441643; 0125441649
• ISSN: 0091-679X
• DOI: 10.1016/S0091-679X(08)61541-1

This chapter focuses on the immunogold labeling technique for transmission electron microscopy. Pre-embedding and post-embedding labeling protocols with a single primary antibody and a double labeling protocol with two different primary antibodies are described. The labeling protocols include a description of a morphometric analysis to quantitate colloidal gold distribution in labeling experiments. Colloidal gold-labeled antisera are widely used to map intracellular distribution of antigens. To use this technique, a primary antibody is bound to antigenic sites in situ, and a secondary antibody, coupled to colloidal gold and specific for the general class of the primary antibody, is bound to the primary antibody. Colloidal gold particles are readily visible as dense spheres in electron microscope images. Colloidal gold labeling has been used to map Tetrahymena proteins in the oral apparatus, membrane skeleton, and nucleolus. For double labeling, two different primary antibodies are used followed by secondary antibodies labeled with gold particles of different size.