Immunogold labeling of flagellar components in situ.
Immunogold electron microscopy is a classic high-resolution method for the selective localization of macromolecules in the context of cells and subcellular structures. Specific antibodies are used to affix small particles of colloidal gold, which are easily visible in the electron microscope, to the macromolecule of interest. There are different immunogold-labeling techniques; in the postembedding immunogold-labeling technique, the biological material is first fixed, dehydrated, and embedded in resin and the antibody reactions are done on the sectioned material. In the preembedding immunogold-labeling technique, the antibody reactions are carried out prior to fixation, dehydration, and resin embedding of the biological specimen. The whole-mount immunogold-labeling technique does not involve resin embedding at all; the material is applied to an electron microscopy grid and the antibody reactions are carried out on the grid. The aim of this chapter is to describe in detail postembedding and preembedding techniques applicable for the immunogold labeling of components of Chlamydomonas flagella and basal bodies. Special emphasis is put on the flat embedding of Chlamydomonas cells, which allows the analysis of individual flagella along their whole length, a method especially suitable to the study of intraflagellar transport (IFT). Depending on the fixation protocol and resin used, such flat embeddings can be utilized for the localization of components of the IFT machinery by postembedding immunogold labeling or the ultrastructural analysis of the IFT complex by standard electron microscopy or electron tomography.
Geimer
Stefan
S
Zellbiologie/Elektronenmikroskopie NWI/B1, Universität Bayreuth, 95440 Bayreuth, Germany.
D000818:Animals; D002696:Chlamydomonas / Q000648:ultrastructure; D003599:Cytoskeleton / Q000648:ultrastructure; D005407:Flagella / Q000648:ultrastructure*; D007150:Immunohistochemistry / Q000295:instrumentation / Q000379:methods*; D008854:Microscopy, Electron / Q000295:instrumentation / Q000379:methods*; D016610:Tissue Embedding / Q000379:methods; D016707:Tissue Fixation / Q000379:methods