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Fluorescent detection is commonly used to visualize multiple antigens simultaneously. The fluorochrome may be conjugated directly to the primary or secondary antibody or to streptavidin.
When designing multicolor experiments, two key parameters must be considered. Firstly, spectral overlap between the fluorochromes being used should be limited as much as possible. Secondly, if indirect detection methods are employed, cross-reactivity between the detection reagents must be avoided (where possible). This is usually achieved by selecting primary antibodies from different species, ensuring that each secondary antibody only recognizes one primary antibody in the experiment.
Two primary antibodies from the same species may be used if one of the primary antibodies is biotinylated. In this method, the tissue is incubated with the non-biotinylated antibody first, followed by incubation with the corresponding flurochrome-conjugated secondary antibody. The tissue is then incubated with the biotinylated antibody, followed by incubation with a streptavidin-conjugated fluorophore, which binds to the biotin conjugated to the primary antibody. This method is susceptible to high background staining due to endogenous biotin, particularly when using frozen tissues.