Methods Immunolabelling

The two most established methods of labeling a molecule or pair of molecules to make a FRET measurement are immunolabeling and fusion to genetically encoded FPs like GFP. Although these are well established techniques, they have certain drawbacks and so novel sensors like QDs and labeling techniques, like cysteine-reactive fluorophores continue to be developed and offer great promise for the future. 

Kanitakis, J. and Thivolet, J. (1987). Immunolabeling methods in cutaneous histopathology. Principles and practical applications. Ann. Pathol. 7,79-97. 

The process of immunolabeling for each of these different methods is similar, The difference between the techniques is in preparation of the antigen and the contrasting method (see Note 3). 

The preembedding technique is used to localize antigens on the surface of isolated cells, either prokaryotes or eukaryotes. If it is necessary to store the cells before immunolabeling, they must be fixed as in Section 3.1.1.1. If a very sensitive method is required, fixation may be omitted before immunolabeling. 

However, researchers should be aware that a new noncovalent method for biotinylating antibodies has recently become available. Zenon technology from Molecular Probes, Inc., is a unique immunolabeling system that enables researchers to attach biotin,... 

Cryostat sections and cytocentrifuge preparations should be air-dried for at least 1 h but preferably overnight before immuno-staining. Before immunolabeling, cryostat sections should be fixed in cold acetone for 10 min and cytospin slides should be fixed for 90 s in a 1 1 mixture of acetone and methanol at room temperature. After fixation, follow the IGSS method for paraffin sections from step 3. Cytospin preparations are usually adequately covered by standard... 

De Waele et al. (9) recommend immunolabeling isolated cells while in suspension. This necessitates centrifugation after each step until a cytocentrifuge preparation is made prior to contrasting. A preferred method in this laboratory is to prepare cytocentrifuge preparations and to immunolabel them as for frozen sections thereby avoiding several centrifugation steps. 

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. 

Fig. 6. Cellular colocalaation of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in the neostriatum. Each number indicates the same glial cell showing both GDH and GS immunoreactivities. The figure is modified from Kaneko et al. (1988a), where the method for double staining is described. Briefly, GS was immunolabeled with anti-GS rabbit serum and fluorescein-labeled anti-rabbit IgG antibody, and after blocking the sections with normal rabbit serum GDH was visualized by the immunoperoxidase method with biotinylated anti-GDH rabbit IgG and ABC.

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