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Antibodies bind stably and specifically to their corresponding antigen, they are invaluable as probes for identifying a particular molecule in cells, tissues, or tissues, or biological fluids. Antibody molecules can be used to locate their target molecules accurately in single cells or in tissue sections by a variety of different labeling techniques. When the antibody itself, or the antiimmunoglobulin antibody used to detect it is labeled with a fluorescent dye the technique is known as immunoflurescence microscopy. As in all serological techniques, the antibody binds stably to its antigen, allowing unbound antibody to be removed by thorough washing. As antibodies to proteins recognize the surface features of the native, folded protein the native structure of the protein being sought usually needs to be preserved, either by using frozen tissue sections that are fixed only after the antibody reaction has been performed. Some antibodies, however, bind proteins even if they are denatured, and such antibodies will bind specifically even to protein in fixed tissue sections.
The fluorescent dye can be covalently attached directly to the specific antibody, but more commonly, the bound antibody is detected by fluorescent anti-immunoglobulin, a technique known as indirect immunofluorescence. The dyes chosen for immunofluroscence are exited by light of one wavelength, usually blue or green, and emit light of a different wavelength in the visible spectrum. The most common fluorescent dyes are fluorescein, which emitsgreen light, Texas Red and Peridinn chlorophyll protein (PerCP), which emit red light, and rhodamine and phycoerythrin (PE) which emit orange/red light. By using selective filters, only the light coming from the dye or flurochrome used is detected in the fluorescence microscope. This technique can be used to detect the distribution of any protein. By attaching different dyes to different antibodies, the distribution of two or more molecules can be determined in the same cell or tissue section.
The recent development of the confocal fluorescent microscope, which uses computer-aided techniques to produce an ultrathin optical section of a cell or tissue, gives very high resolution immunofluorescence microscopy without the need for elaborate sample preparation. The resolution of the confocal microscope can be further increased using low-intensity illumination so that two photons are required to excite the flurochrome. A pulsed laser beam is used, and only when it is focused into the focal plane of the microscopeis the intensity sufficient to excite fluorescence. In this way the fluorescence emission itself can be restricted to the optical section.
One important development in the area of microscopy has been the use of time-lapse video microscopy, in which sensitive digital video cameras record the movement of fluorescently labeled molecules in cell membranes and their redistribution when cells come into contact with each other. Cell-surface molecules can be fluorescently labeled in two main ways. One is by the binding of flurochrome-labeled Fab fragments of antibodies specific for the protein of intrest; the other is by generating a fusion protein, in which the protein of intrest has been attached to one of a family of florescent proteins obtained from jellyfish like GFP. The list of available fluorescent labels now includes red, blue, cyan or yellw fluorescent proteins.