Streptavidin fluorochrome labeling

When one or more of the desired antibodies is not available as a direct conjugate and a conjugate cannot be synthesized, indirect methods have to be used. The simplest variation is to use a biotin-labeled antibody as a first layer, and then after washing the cells, incubate with fluorochrome-labeled streptavidin, and additional directly labeled antibodies labeled with complementary fluoro-chromes. The additional streptavidin step does not usually cause difficulty... 

Fluorochrome labeling of streptavidin or antibody Conjugation procedures should yield optimal fluorochrome/protein (F/P) ratios. Most economically, the desired F/P ratio is regulated by the initial weight of dye in the reaction mixture and the reaction is allowed to go to completion. Alternatively, with relatively more dye, the reaction is interrupted after a specific incubation period. The efficiency of labeling depends on the protein, the protein concentration, the specific fluorochrome and the purity of the fluorochrome (some preparations only 30%). Over- or undercoupling leads to nonspecificity or low detectability, respectively. 

Fig. 1 Diagram illustrating the molecular interactions of direct immunofluorescent labeling method. The fluorochrome-labeled antibody reacts with the cell or tissue antigen in a one-step staining procedure (a). Alternatively, the primary antibody, labeled with biotin, can be used with a secondary fluorochrome-streptavidin reagent to increase signal strength (b).

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