11/24/2014

Immunofluorescence Protocol | Immunohistochemistry Protocol | Immunofluorescence Techniques| immunofluorescence staining protocol | Acade

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Immunofluorescence
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by Jean-Marc Fritschy, Wolfgang Hrtig

Introduction
Immunofluorescence is one of many techniques in biomedical research and diagnostics that makes use of the sensitivity and
selectivity of fluorescence for analysing biological tissues. Its main application is the detection of proteins and other biomolecules in
cells and tissues. Closely related techniques include the use of fluorescent reporter gene products, such as green fluorescent protein
(GFP), for probing gene expression, tracing cell lineage or as fusion tags to monitor protein localization within living cells, as well as
fluorescence in situ hybridization (FISH), to detect nucleic acids (DNA and RNA) with fluorochromated nucleotide probes. Another
important application of fluorescence in tissues is ion (ratio) imaging, which takes advantage of fluorescent probes sensitive to the
concentration of specific ions (e.g. calcium) to monitor their concentration in the cytoplasm and organelles of single living cells. See
also Green Fluorescent Protein (GFP), Fluorescence in situ Hybridization, and Fluorescent Probes Used for Measuring Intracellular
Calcium
Immunofluorescence is based on the high selectivity and affinity of antibodies for their antigens as specific cellular constituents, notably proteins. Antibodies are typically
raised against purified antigen preparations, recombinant proteins, or synthetic peptides coupled to a carrier protein. Optimal detection depends on the quality of the
antibodies (high titre and affinity), the preservation of the tissue or cells to be examined, the sensitivity of the assay, the quantum yield and photostability of the fluorochrome,
and the lack of autofluorescence. Equally important is the quality of the detection devices (fluorescence microscope, flow cytometer), which will also be reviewed in this article.
The sensitivity of immunofluorescence can be greatly improved by increasing the number of fluorescent molecules per antigen to be detected. This is usually achieved by
indirect immunofluorescence (Figure 1). With this technique, the first (primary) antibodies directed against the target structures or molecules are unlabelled. They are then
bound by secondary antibodies raised against immunoglobulins of the host species used for the primary antibodies. The secondary antibodies are labelled either with
fluorochromes or with other haptens (e.g. biotin, digoxin), which serve as anchoring sites for enzymes or fluorescent molecules, or as targets for a third antibody. Since the
primary antibodies can bind more than one secondary antibody, the signal is amplified. Furthermore, this approach provides considerable versatility, since a given primary
antibody can be used in combination with innumerable secondary antibodies and detection procedures.

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