Use of antibodies in fluorescence microscopy

Antibodies for immunofluorescence are divided into two groups: primary and secondary antibodies. Depending on the selected technique only a primary, or a primary and secondary are used:

• Direct fluorescence: Uses only primary antibodies, which bind the epitope and are directly conjugated with a fluorophore. It requires a simpler procedure and, since secondary antibodies are not needed, it avoids any cross-reactivity between them. However, the procedure is normally more expensive and difficult if there are not enough conjugates available for different colors required, as the fluorescent signal depends on the number of fluorophores that can be attached, limiting the detection of highly abundant epitopes.

• Indirect fluorescence: Uses both primary and secondary antibodies to label the target. Primary antibodies are normally monoclonal and have a high specificity for the targeted epitope in the protein, while secondary antibodies are normally polyclonal, conjugated with fluorophores, and recognize different epitopes of the primary antibody in order to increase the fluorescence signal. In addition to the increased signal, production of secondary antibodies is relatively cheap and there is a broad range of available colors, which allows more flexibility in the experiment. It's important to select antibodies that were generated in different species to avoid any cross-reactivity, taking into account that the secondary antibody should have been raised against the same species as the one the primary was generated in.