Immuno-Fluorescence (IF) is a technique of immunostaining which means that it uses antibodies' affinity for their antigens to dye their target location. IF uses the fluorescence phenomenon to make antigens visible. Fluorescent dyes, or fluorophore, are conjugated to antibodies that will bind to their antigen, thus revealing their localization during a microscope observation. Immunofluorescence is especially used on fixed dead cells to localize the antigens inside it.
Fluorescence is a property of some molecules that get excited and absorb light at a specific wavelength and reemit this energy as heat and light at another specific wavelength. The wavelengths, and thus colors, of absorption and emissions depend on the molecule. Therefore a polychromatic light can excite different fluorophores at the same time which will emit each in their own wavelength. In immunofluorescence, this property can be used to color different antigens at the same time.
When conducting an Immunofluorescence experiment, researchers have to choose carefully the fluorophores. One of the main considerations is photobleaching or the fading of the color that has to be as lower as possible during the experiment.
In Primary (or Direct) Immunofluorescence a primary antibody is conjugated to the fluorophore. This first type of IF is less used than the other one. The main limitation on this technique is that only one antibody can bind to one antigen. It is therefore not very sensitive. On the other hand, using a single primary antibody reduces background signal and cross-reactivity.
In Secondary (or Indirect) Immunofluorescence the primary does not bear any fluorophore. The dye is conjugated to a secondary antibody that targets the constant region of the primary antibody. This system provides an amplification of the fluorescent signal as several secondary antibodies may be able to target one primary antibody. It enables also the use of a wider range of detections.