Immunofluorescence is widely used on tissue sections, cultured cell lines, along with individual cells. It also helps in analyzing the distribution of proteins, glycans, biological and non-biological molecules. It is also great in the visualization of intermediate sized filaments.

There are two types of immunofluorscence techniques, direct or primary, and indirect or secondary.

Direct or primary immunofluorescence technique employs a single antibody which is linked to a fluorophore chemically. This antibody first recognizes the target molecule and then binds to it. The fluorophore it is carrying will get detected by microscopy. There are several advantages of Direct immunofluorescence over Indirect because of the direct connection of the antibody to the fluorophore. This reduces the staining procedure steps which makes the process faster. It also reduces the background signal by avoiding particular issues with antibody cross-reactivity. Still, Direct immunofluorescence is less sensitive than Indirect immunofluorescence as the number of fluorescent molecules which can bind to the primary antibody is limited.

Indirect or Secondary immunofluorescence employs two antibodies. One is a primary unlabeled first antibody. It specifically binds the target molecule. Another is the secondary antibody which carries the fluorophore. It also recognizes the antibody and binds to it. In this fluorescence, multiple secondary antibodies can bind to the primary ones. As the number of fluorophore molecules per antigen increases, it results in signal amplification. Indirect immunofluorescence is more complex and time-consuming than direct immunofluorescence, but there is more flexibility in this process as there are varied secondary antibodies and detection techniques can be used for a given primary antibody.