Immunofluorescence is a technique used to detect the expression of specific proteins in cultured cells or on tissue sections. It is widely used in molecular biology. This technique is based on the principle of immunoreaction, in which specific antibodies binds to specific antigens. In immunofluorescence, the protein that needs to be detected is treated as a target "antigen", and a designed antibody (normally an immunoglobulin) produced by a different animal species, specifically against the antigen will then be applied on the cell or tissue section. This antibody is labelled with a fluorescent dye and will bind to the target protein. The fluorescent dye could give a fluorescent signal if exposed to laser or other exciting light. The fluorescent signal can be visually seen under a microscope. Microscopes with the exciting light are called fluorescence microscope and can be used to capture images with the fluorescent signal for later analysis. In general, a stronger fluorescent signal means a stronger expression of the target protein in the cells or on the tissue, because the more the protein is expressed the more antibody bind to the sample and thus more fluorescent dye labels. However, the fluorescent signal might fade gradually during exposure, so images should be taken as quickly as possible.
In some cases, the fluorescent dye is not directly labelled on the antibody that binds to the target protein, but on a "secondary" antibody from a third species that specifically binds to the first antibody. This double selective binding can increase the immunofluorescence specificity.
- ↑ Donaldson JG. Immunofluorescence staining. Current protocols in cell biology. 2001 May; Chapter 4: Unit 4 3. PubMed PMID: 18228363. Pubmed Central PMCID: 4709840.