IMMUNOELECTRON MICROSCOPY

The purpose of this chapter is to provide a practical introduction to the technique of immunoelectron microscopy that is a tool for the ultrastructural localization of flagellar/ciliary components. As polyclonal antibodies recognize multiple antigenic determinants per target molecule, they generally produce better signals than monoclonal antibodies. Antibody specificity is to be evaluated by immunoblot analysis. There is detailing of the specimen preparation involving embedding and thin sectioning, embedment of Chlamydomonas in L.R. white, flagellar whole mounts, and tips for incubating EM grids on various solutions. Frozen thin sections provide good membrane preservation useful for the analysis of flagellar membrane-associated antigens. Freed from the masking effects of heavy fixation, dehydration, and embedment, axonemal whole mounts show excellent labeling densities. There is also a description of the method for the preparation of Chlamydomonas axonemal whole mounts. There are tips for incubating EM grids on various solutions. Successful immunolabeling of material on EM grids requires that the same grid be handled many times without damage. One way to accomplish this is to use a modified inoculating loop to move grids from solution-to-solution. Also a description of immunolabeling technique has been provided. Details have been presented on the choice of gold marker and Immunogold labeling of specimens on EM grids. In the absence of poststaining, the gold particles stand out well, to evaluate levels of specific staining of structures, and of background labeling. The initial screening of antibodies by immunofluorescence indicates where antigen should be detected. An ideal control is provided by performing identical localizations on mutant cells that lack the specific gene product being localized. In evaluation of antisera in immunofluorescence, a positive signal provides a guide, indicating where antigen is localized and suggesting that the antigen is concentrated enough to be detected at the EM level. © 1995, Academic Press Inc.