Information AboutImmunofluorescence |
| CATEGORIES ABOUT IMMUNOFLUORESCENCE | |
| immunology | |
| biological techniques and tools | |
| anatomical pathology | |
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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 Fluor s or DyLight Fluor s). Many uses of immunofluorescence have been outmoded by the development of Recombinant Protein s 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. EXTERNAL LINKS
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