33.15: Immunogold Electron Microscopy
Immunoelectron microscopy utilizes immunogold labeling of endogenous proteins with specific antibodies to detect and localize these proteins in cells and tissues. The procedure provides insights into the distribution and quantification of protein under different stimulation conditions offering clues about their functions. Conjugating highly electron-dense gold particles with primary or secondary antibodies allow antigen detection on and within cells, with high resolution and specificity. Immunoelectron microscopy has been used to identify the cellular and subcellular localization of proteins involved in neurotransmission, nuclear protein components, and identification of immune cell types.
Three approaches are applied to localize cell antigens using transmission electron microscopy. First, when studying the localization of intracellular antigens, protocols involve antibody labeling post-embedding in acrylic resins, antibody labeling pre-embedding in the resin combined with cell membrane permeabilization, or cryo-ultramicrotomy without embedding. Second, when cell-surface proteins are to be localized, the pre-embedding protocol is used where labeling with immunogold antibodies is done before embedding in the resin. The third technique, the whole-mount technique, does not involve resin-embedding, and the antibody reactions are carried out directly on a sample to locate the surface molecules.
For scanning electron microscopy, the surface of interest must necessarily be exposed. The sample can be directly fixed and labeled if the outer surface of the plasma membrane is of interest. However, the cells must be permeabilized with detergents and immunolabeled for studying intracellular components or antigens integrated into a tissue. For viewing, the sample is freeze-fractured (rapid freezing and breaking with a knife). Many intracellular components, particularly macromolecular complexes, like chromatin, protein complexes, or viruses, can be isolated, fixed, and immunolabeled for SEM. Finally, an immunonegative staining technique is applied for surface antigens on small specimens, such as viruses and bacteria, which lend themselves to negative staining.