Ultrastructural Localization of Peptides Using Immunogold Labeling

The subcellular localization of bioactive peptides, including hormones and neurotransmitters, has immense value, not only in understanding how cells function, but in correlating biochemical and clinical data from tissues in both normal and diseased states. The pioneering immunocytochemical work of Coons et al. (1 ) exploited the specificity of antigen-antibody interactions by using fluorescent-labeled antibodies as probes to determine the spatial distribution of antigens. It established the principles of immunocytochemistry, and thereby revolutionized the means of identifying peptide-containing cells by light microscopy. The application of immunocytochemistry at the ultrastructural level combines the specificity and sensitivity it offers with the fine spatial definition of electron microscopy, providing an exceedingly precise means of localizing the position of specific peptide antigens to a resolution of 5 nm or better. As a technique, electron microscopic immunocytochemistry was first accomplished some 35 yr ago (2 , with the development of ferritin-antibody conjugates. Since then, the introduction of colloidal gold (3 ) as a tracer in electron microscopy, and its use as a marker for antisera (4 ), has established immunogold labeling as by far the most popular choice for ultrastructural localization of bioactive peptides and most other immunogens. This is not surprising since gold spheres are easily prepared in the laboratory, and are readily adsorbed by a variety of proteins, including immunoglobulins, with which they form simple but stable antibody-gold complexes without involving chemical conjugation.