Multiple immunolabeling

Beesley JE. Multiple immunolabeling techniques, mlmmunocytochemistry, APrac-tical Approach (Beesley JE, ed.), Oxford University Press, Oxford, UK, 1993, pp. 103-125. 

The protocol for double/multiple immunolabeling using haptenylated primary antibodies is essentially the same as with primary antibodies of different IgG isotypes. These protocols can be easily customized depending on the availability of primary antibodies for your research requirements. For instance, you may have at your disposal a pair of monoclonal antibodies of the same IgG isotype, and only one of them is haptenylated. In this case, you have to carry out the immunostaining in two steps in the first step you visualize the unlabeled first primary antibody with a secondary species-specific antibody, and in the second step you can detect the second primary haptenylated antibody via another secondary antibody directed against the corresponding hapten. Should the hapten be a fluorophore, it can be visualized directly in a fluorescent microscope and you do not need the second step... 

The nominal values for the sizes of the gold are those given above. In practice, these tend to vary slightly. Before the gold probes are used for electron microscopy, it is advisable to check the size range, especially if multiple immunolabeling is to be carried out. 

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. 

Pujic, Z., Savage, N. W., Bartold, P. M., and Walsh, L. J. 1998. Simultaneous detection of multiple antigens with triple immunolabeling. Appl. Immunohistochem. 6 150-153. 

We routinely silanate glass microscope slides that have small wells 3-5 mm in diameter that have been formed by a paint coating. These are available commercially (not silanated) from Roboz (Rockville, MD). Alternatively, cut holes in strips of vinyl electrical insulation tape using a hole punch and apply this tape to microscope slides that have previously been silanated. This tape forms small wells that contain the drops of immunoreagents that are subsequently applied. The use of small wells conserves expensive immunoreagents and allows multiple dilutions of an antibody to be tested on different wells of a single slide. Sections of the tissues are cut and deposited onto the slides. The sections are allowed to air dry at room temperature for 20-30 min prior to commencement of immunolabeling... 

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