Antibodies are proteins produced by the vertebrate immune system as a defense against infection (discussed in Chapter 24). They are unique among proteins in that they are made in billions of different forms, each with a different binding site that recognizes a specific target molecule (or antigen). The precise antigen specificity of antibodies makes them powerful tools for the cell biologist. When labeled with fluorescent dyes, antibodies are invaluable for locating specific molecules in cells by fluorescence microscopy (Figure 9–16); labeled with electrondense particles such as colloidal gold spheres, they are used for similar purposes in the electron microscope (discussed below). The antibodies employed in microscopy are commonly either purified from antiserum so as to remove all nonspecific antibodies, or they are specific monoclonal antibodies that only recognize the target molecule.
When we use antibodies as probes to detect and assay specific molecules in cells, we frequently use chemical methods to amplify the fluorescent signal they produce. For example, although a marker molecule such as a fluorescent dye can be linked directly to an antibody—the primary antibody—a stronger signal is achieved by using an unlabeled primary antibody and then detecting it with a group of labeled secondary antibodies that bind to it (Figure 9–17). This process is called indirect immunocytochemistry.
Some amplification methods use an enzyme as a marker molecule attached to the secondary antibody. The enzyme alkaline phosphatase, for example, in the presence of appropriate chemicals, produces inorganic phosphate that in turn primary antibody: secondary antibodies:
Figure 9–16 Immunofluorescence.
a transmission electron micrograph of the periphery of a cultured epithelial cell showing the distribution of microtubules and other filaments. (B) the same area stained with fluorescent antibodies against tubulin, the protein that assembles to form microtubules, using the technique of indirect immunocytochemistry (see Figure 9–17). Red arrows indicate individual microtubules that are readily recognizable in both images. note that, because of diffraction effects, the microtubules in the light microscope appear 0.2 μm wide rather than their true width of 0.025 μm. (From M. osborn, r. Webster and k.
Weber, J. Cell Biol. 77:r27–r34,  1978. With permission from the rockefeller University press.)
Figure 9–17 Indirect immunocytochemistry.
this detection method is very sensitive because many molecules of the secondary antibody recognize each primary antibody. the secondary antibody is covalently coupled to a marker molecule that makes it readily detectable. Commonly used marker molecules include fluorescent probes (for fluorescence microscopy), the enzyme horseradish peroxidase (for either conventional light microscopy or electron microscopy), colloidal gold spheres (for electron microscopy), and the enzymes alkaline phosphatase or peroxidase (for biochemical detection).
leads to the local formation of a colored precipitate. This reveals the location of the secondary antibody and hence the location of the antibody–antigen complex. Since each enzyme molecule acts catalytically to generate many thousands of molecules of product, even tiny amounts of antigen can be detected. Although the enzyme amplification makes enzymelinked methods sensitive, diffusion of the colored precipitate away from the enzyme limits the spatial resolution of this method for microscopy, and fluorescent labels are usually used for the most sensitive and precise optical localization.
imaging of Complex three-Dimensional objects is possible with the optical Microscope