33.15:
Immunogold Electron Microscopy
In immunogold electron microscopy, a target molecule is labeled using gold-conjugated antibodies.
The gold increases the electron scattering to give high-contrast dark spots. These spots can be distinguished from the other unlabelled structures and allow localization of the target molecule.
Direct immunolabeling uses a gold probe attached to a primary antibody that directly binds to the target molecule.
Indirect labeling uses a gold-labeled secondary antibody that binds to an unlabelled primary antibody. Indirect labeling is used more often due to its higher sensitivity.
Immunolabeling can be performed using several different techniques — post-embedding, pre-embedding, or whole-mount.
In the post-embedding technique, a sample is embedded in resin and then sliced into ultrathin sections that are then treated with gold-labeled antibodies.
In the pre-embedding technique, a sample is first incubated with the gold-labeled antibodies and then resin-embedded and cross-sectioned.
The whole-mount technique does not involve resin-embedding. Antibody reactions are carried out directly on a sample placed on the sample holder.
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.