Immunofluorescence

Immunofluorescence

Immunofluorescence is the labeling of antibodies or antigens with fluorescent dyes. This technique is often used to visualize the subcellular distribution of biomolecules of interest. Immunofluorescent-labelled tissue sections or cultures are studied using a fluorescence microscope or by confocal microscopy.

Most commonly, immunofluorescence employs two sets of antibodies: a primary antibody is used against the antigen of interest; a subsequent, secondary, dye-coupled antibody is introduced that recognizes the primary antibody. In this fashion the researcher may create several primary antibodies that recognize various antigens, but, because they all share a common constant region, may be recognized by a single dye-coupled antibody. Typically this is done by using antibodies made in different species. For example, a researcher might create antibodies in a goat that recognize several antigens, and then employ dye-coupled rabbit antibodies that recognize the goat antibody constant region (denoted rabbit anti-goat). This allows re-use of the difficult-to-make dye-coupled antibodies in multiple experiments.

In some cases, it is advantageous to use primary antibodies directly labelled with a fluorophore. This direct labelling decreases the number of steps in the staining procedure and, more importantly, often avoids cross-reactivity and high background problems. Fluorescent labelling can be performed in less than one hour with readily available labeling kits.

As with most fluorescence techniques, a significant problem with immunofluorescence is photobleaching. Loss of activity caused by photobleaching can be controlled by reducing the intensity or time-span of light exposure, by increasing the concentration of fluorophores, or by employing more robust fluorophores that are less prone to bleaching (e.g. Alexa Fluors or DyLight Fluors).

Many uses of immunofluorescence have been outmoded by the development of recombinant proteins containing fluorescent protein domains, e.g. green fluorescent protein (GFP). Use of such "tagged" proteins allows much better localization and less disruption of protein function.

Protocol

Immunostaining protocol

External links

* [http://www.ii.bham.ac.uk/clinicalimmunology/CISimagelibrary/ Images associated with autoimmune diseases] at University of Birmingham
* [http://www.confocal-microscopy.org/Protocols%20-%20Immunofluorescence.htm Immunofluorescence protocol and confocal microscopy resources] at confocal-microscopy.org
* [http://www.bio.davidson.edu/COURSES/genomics/method/IMF.html Overview] at Davidson College
* [http://www.prosci-inc.com/Immunofluorescence-Protocol.html Immunofluorescence Staining Protocol]

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  • immunofluorescence — [ imynoflyɔresɑ̃s ] n. f. • 1965; de immuno et fluorescence ♦ Biol. Technique utilisant un marquage fluorescent d anticorps spécifiques afin de visualiser et de localiser les antigènes correspondants. ● immunofluorescence nom féminin Technique de …   Encyclopédie Universelle

  • immunofluorescence — [im΄yə nōflô res′əns, i myo͞o΄nōflô res′əns] n. the use of a special microscope with ultraviolet light and antibodies labeled with fluorescent dyes to locate specific antigens in tissues immunofluorescent adj …   English World dictionary

  • Immunofluorescence — L’immunofluorescence est une technique d’immunomarquage, qui utilise des anticorps (ou immunoglobulines) ainsi que des fluorochromes. Sommaire 1 Historique 2 Principes de l immunofluorescence[1],[2],[3]. 2.1 Immunofluorescence …   Wikipédia en Français

  • immunofluorescence — An immunohistochemical technique using labeling of antibodies by a fluorescent dye to identify antigenic material specific for the labeled antibody; the specific binding of antibody can be determined microscopically through the production of a… …   Medical dictionary

  • immunofluorescence — n. a technique for observing the amount and/or distribution of antibody or antigen in a tissue section. The antibodies are labelled (directly or indirectly) with a fluorescent dye (e.g. fluorescein) and applied to the tissue, which is observed… …   The new mediacal dictionary

  • immunofluorescence assay — Med. a diagnostic blood test using the technique of immunofluorescence. Abbr.: IFA * * * …   Universalium

  • immunofluorescence microscopy — fluorescence microscopy using immunofluorescence (q.v.) staining methods …   Medical dictionary

  • immunofluorescence assay — Med. a diagnostic blood test using the technique of immunofluorescence. Abbr.: IFA …   Useful english dictionary

  • immunofluorescence — noun Date: 1960 the labeling of antibodies or antigens with fluorescent dyes especially for the purpose of demonstrating the presence of a particular antigen or antibody in a tissue preparation or smear • immunofluorescent adjective …   New Collegiate Dictionary

  • immunofluorescence — (im u no floo o res ens) A technique used to identify particular antigens microscopically in cells or tissues by the binding of a fluorescent antibody conjugate …   Dictionary of microbiology

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