Immunofluorescence

Immunofluorescence is a technique that takes advantage of the specific antibody-antigen binding to target fluorophores to biomolecules in the cell. Fluorophores are attached, or conjugated, to antibodies (Figure 1). These fluorescently labeled antibodies may bind to the target directly (primary antibodies) or indirectly (secondary antibodies). Secondary immunofluorescence uses a non-fluorescent primary antibody that binds to the target and a fluorescently tagged secondary antibody that binds to the primary antibody.

Figure 1: Fluorescently labeled antibodies binding to a molecule of interest either directly (left, primary antibody only) or indirectly (right, primary and secondary antibody).

While more time consuming, secondary antibodies allow for more flexibility. The same primary antibody could be labeled with different colors, for example. Because multiple secondary antibodies can bind to a single primary antibody, the signal can also be amplified.