Immunolabeling colloidal gold

Immunohistochemical labeling for electron microscopy is based on the same principles as immunohistochemistry for light microscopy. The differences are that specimen sections must be much thinner (50 100 nm) and the label must be electron-dense. The first electron-dense labels used for immunolabeling at the electron microscope level were ferritin and peroxidase. Peroxidase label can be visualized using DAB reaction product which becomes electron-dense after osmi-cation. With the advent of colloidal gold particles as markers in immunocytochemical... 

The immunoreplica technique (14) is used when it is necessary to detect antigenic sites on the plasma membrane of cultured cells. The cells are cultured on coverslips, and are fixed as described above depending on the antibody in question, and immunolabeled in situ as described in Section 3.1.1.2., steps 3-9. After immunolabeling (Section 3.1.1.2., step 9), they are further fixed with 1% osmium tetroxide and are dehydrated in a graded series of ethanol (70, 90, 100%), critically point-dried, and replicated with a layer of carbon and platinum, The replicas are cleaned with sodium hypochlorite and chronic acid before examination with the transmission electron microscope. Large areas of the replicated plasma membrane remain intact for observation. Colloidal gold probes are probably the only probes of sufficient density that can be detected on these surfaces. 

Colloidal gold probes are the most popular of all the immunolabeling techniques for ultrastructural immunocytochemistry and in situ hybridization Since individual gold probes can be easily identified with the electron microscope, there is... 

Figure 14.5 SNMP immunolabeling of EM sections of pheromone-sensitive trichoid sensilla. SNMP antibodies were visualized using secondary antibody conjugated to 10 nm colloidal gold particles. A and B include sensillum cuticle C and D show only dendrites. Sensilla contained two neurons one neuron consistently showed significantly greater labeling. Scale bar. 1.25 pm (A),...

Until 1980, peroxidase was the marker of choice, but since then, the use of colloidal gold has increased and now, is almost universally used for electron immunocytochemistry Colloidal gold is ideal for electron microscopy. It is particulate tind very dense, therefore, it can be identified on heavily stained biological tissue. Because it is small, it will not obscure the fine structure of the sample, and it can be prepared in several different sizes to be used for multiple immunolabeling experiments and quantification. 

Colloidal gold probes are the most popular of all the immunolabeling techniques for electron immunocytochemistry. Since individual gold probes ctin be easily identified with the electron microscope, there is increased interest in muldple immunolabeling and quandtative studies. The technique is so popular that there are many different nuances in immunolabeling technique. The methods given in this chapter are those that have proved to be sadsfactory in this laboratory. 

Y-D. Stierhof, H. Schwarz, M. Durrenberger, W. Villiger, and E. Kellenberger, Yield of Immunolabel Compared to Resin Sections and Thawed Cryosections, in Colloidal Gold Principles, Methods and Applications, Vol. 3 (ed. M. A. Hayat), Academic Press, New York, 1991, pp. 87-95. 

Armbruster, B. L., Garavito, R. M., and Kellenberger, E. (1983). Dehydration and embedding temperature affect the antigenic specificity of tubulin and immunolabelling by the protein A colloidal gold technique. J. Histochem. Cytochem. 31, 1380-1384. 

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