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

• Direct fluorescence:Uses only primary antibodies, which bind theepitopeand are directly conjugated with afluorophore. 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 normallymonoclonaland have a high specificity for the targeted epitope in the protein, while secondary antibodies are normallypolyclonal, 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.