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Encyclopedia > Immunofluorescence

Immunofluorescence is the labeling of antibodies or antigens with fluorescent dyes. This technique is sometimes used to make viral plaques more readily visible to the human eye. Immunofluorescent labelled tissue sections are studied using a fluorescence microscope or by confocal microscopy. Schematic of antibody binding to an antigen An antibody or immunoglobulin is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. ... An antigen is any molecule that is recognized by antibodies. ... Fluorescence induced by exposure to ultraviolet light in vials containing various sized Cadmium selenide (CdSe) quantum dots. ... Yarn drying after being dyed in the early American tradition, at Conner Prairie living history museum. ... A viral plaque is a visible structure formed within a cell culture, such as bacterial cultures within some nutrient medium (e. ... Confocal laser scanning microscopy (CLSM or LSCM) is a valuable tool for obtaining high resolution images and 3-D reconstructions. ...


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.


As with most fluorescence techniques, a significant problem with immunofluorescence is photobleaching. Photobleaching is the destruction of a photochemical fluor by high-intensity light. ...


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. Recombinant proteins are proteins that are produced by different genetically modified organisms following insertion of the relevant DNA into their genome. ... GFP ribbon diagram from PDB database The green fluorescent protein (GFP) is a protein from the jellyfish Aequorea victoria that fluoresces green when exposed to blue light. ...


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  Results from FactBites:
 
Dang, Mol Vis 2004; 10:323-327. (2900 words)
Results: Punctate immunofluorescence, indicating the presence of gap junctions, was observed in the inner and outer plexiform layers of both wild type and cone-less and rod-less retinas.
Immunofluorescence analysis of connexin 36 in fixed retinas using a connexin 36 specific antibody showed that the expression of the protein is greatly increased in the OPL of cone-only retina in comparison to the control and that it is marginally lower in the absence of cones.
Densitometric analysis of the immunofluorescence, shown in Table 1, revealed the OPL of the cone-only retina to be 2050% of that of the wild type.
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