Fluorescent detection (immunofluorescence) methods use fluorochrome labels, which emit light of a longer wavelength when excited by light of a specific wavelength.
Using Immunofluorescence in Antigen Analysis
Immunofluorescence, i.e. detection of antigens via fluorescence, can be used to observe several different antigens in one experiment. Immunofluorescence uses fluorochrome labels attached to antibodies, when the labels are excited by a specific wavelength of light they begin to emit light of a longer wavelength, allowing the presence and position of antigens in a sample to be determined.
Two important parameters must be taken into account when running a multi color experiment:
- If an indirect detection method is used, i.e. secondary antibodies, then the cross-reactivity between different detection reagents must be considered, and, if possible, avoided. This can be ensured by using primary antibodies sourced from different species which means that each secondary antibody can only recognize one of the antibodies in the experiment.
- The spectral output of the two different fluorochromes should have minimal overlap.
Labeling Primary Antibodies
If one of the primary antibodies in the experiment is labeled via biotinylation then two primary antibodies sourced from the same species can be used. To do this, the tissue should first be incubated with the non-biotinylated antibody and then by incubation with the non-biotinylated antibody’s fluorochrome tagged secondary antibody.
Following the first sequence of incubations, the tissue should then be incubated with the biotinylated antibody and then incubated with a fluorophore conjugated to streptavidin. The streptavidin-conjugated fluorophore then binds to the primary antibody by binding to its biotin tag.
However, care must be taken as this method can result in a high level of background staining as a result of biotin’s endogenous nature. This is particularly prevalent when frozen tissues are used.
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