Immunolabeling techniques

Immunolabeling techniques for transmission electron microscopy can be divided into three basic categories: no embedding, pre-embedding and postembed-ding. The no-embedding method is used primarily for immunostaining large biological molecules, such as proteins and nucleic acid-protein complexes, viruses, and frozen cryostat sections mounted on film-coated grids that need no further fixation or intramolecular supporting matrix. The pre-embedding technique is used for labeling cell surfaces (see Figures 1 and 2). This technique does not readily permit access to intracellular antigens. In the pre-embedding technique, unfixed or mildly fixed material is immunostained before the embedding and sectioning processes. In the postembedding method, the material is lightly fixed, embedded and thin sectioned. The thin sections are then immunostained. Figure 4 shows an application of the postembedding method in localizing an intracellular protein. This methodology is the most versatile in visualizing intracellular structures and associated antigens simultaneously and has overcome the many problems incurred by other methods. Since mild fixation and embedding are required in the postembedding thin-section immunostaining method, it should be noted that a number of potential problems can be encountered, including a reduction in immunogenicity, accessibility and concentration of target antigen, during the embedding process, and a loss of ultra-structural definition due to the mild fixation procedures.

All immunolabeling techniques for scanning electron microscopy are performed before dehydration and application of a conductive coating to reveal sur-

Marker conjugated primary Ab

Market conjugated Fab frugmont

Renalured hybrid F<ab)? molecule m

Marker conjugated secondary Ab

Marke* conjugated proleîn A

Maiker conjugated sLrûptav din

Unlabeled primary Ab

Maiker conjugated sLrûptav din

Marker conjugated streplnvidini

Mnrter

Biotinylated secondary Ab

Biotinylated secondary Ab

Unlabeled lenwry Afc

Unlabeled primary

Unlabeled lenwry Afc

Unlabeled sccondnry Ab
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