Immunofluorescence, a common histology labeling technique, relies on antibodies and their attached fluorescent tags to produce images by the matched excitation and emission parameters to the fluorophore of interest. These antibodies are categorized into three main sections: primary antibodies, secondary antibodies, and directly conjugated (DC) antibodies. The proper selection of antibodies is critical to producing clear, informative images for any research project or question. Primary antibodies do not have a fluorescent tag and are paired with secondary antibodies matching the primary host, containing a tag to produce fluorescent signals. This is also called indirect immunofluorescence. In comparison, DC antibodies are primary antibodies with attached fluorescent tags, preventing the need for a secondary antibody, known as direct immunofluorescence.
Developing an antibody panel for immunofluorescence requires the matching of antibody to antigen, as well as the matching of primary and secondary antibodies. Antibodies should be chosen based on wavelength of fluorescence, host, and species reactivity. The correctly chosen primary or DC antibodies should target the antigen of interest, and each antibody chosen for the panel should have a unique host. The correctly chosen secondary antibody should target the host Ig of the primary antibody. Putting two antibodies from the same host species in a panel causes cross-reactivity and false labeling, leading to discrepancies in data.
In addition to choosing antibodies based on reactivity, antibodies should also be chosen based on the wavelength in which fluorescence is visible. DC antibodies and secondary antibodies are only visible at specific wavelengths dependent on fluorophore structure. Only one antibody should be chosen per wavelength or channel, if two antibodies of the same wavelength are chosen, the results and representative images will be incorrect, as the difference between the two antibodies will be indistinguishable.