Immunofluorescence Table of Contents
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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 labeling decreases the number of steps in the staining procedure and, more importantly, often avoids cross-reactivity and high background problems. Fluorescent labeling can be performed in less than one hour with readily available labeling kits.
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.
Applications of Immunofluorescence include:
- tagging proteins
Disadvantages with Immunofluorescence
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).
Because they contain ingredients which act upon each other, many mixtures are most efficient when made up fresh.