Cellular antigens subcellular localization

Immunofluorescence staining permits detection of protein antigens in situ, in order to investigate the subcellular localization or cellular distribution within a tissue. The cells or tissue sections are fixed and incubated with the specific primary monoclonal antibody. The antigen-primaiy monoclonal antibody complex is bound by a second antibody conjugated to a fluorescent dye, such as rhodamine-p-isothiocyanate or fluorescein isothiocyanate, for detection by fluorescence microscopy. Immunofluorescence staining is described in more detail in Chapter 16. 

The membrane is a delicate yet sturdy structure which must be studied intact but cannot be isolated without disruption. In essence, membrane isolation involves the removal of all other parts of the cell. The purity of a membrane preparation is usually measured by the relative increase in specific activity of a membrane-boimd marker enzyme in the membrane preparation over the specific activity of the marker enzyme in the homogenate. In cases where specific membrane markers are lacking, enzymatic activities associated exclusively or primarily with other subcellular components must be demonstrated to be absent in order to show the preparation free from cellular contaminants. Such data, combined with phase-contrast and electron microscopic images of the preparation, are usually accepted as adequate criteria for the measurement of purity. One additional technique, at least for membranes with antigenic properties, is preparation of fluorescent antibodies to the isolated membranes and observation of the antibody localization in whole cells (Kabat and Meyer, 1971). The use of fluorescent probes in determining membrane structure and function has been reviewed (Radda, 1975 Azzi, 1975). 

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