Fluorescence Microscopy in Life Sciences


Author(s): Juan Carlos Stockert and Alfonso Blazquez-Castro

Pp: 462-480 (19)

DOI: 10.2174/9781681085180117010018

* (Excluding Mailing and Handling)


In recent years fluorescence techniques have become increasingly popular for the estimation and localization of enzymatic activities. Basic methodological concepts on enzyme histochemistry can be found in standard books and reviews [1 - 6]. Enzyme activity studies may be carried out to assess the localization of enzymes within cells and tissues, and to determine the effects of pH, ionic strength, temperature, presence of inhibitors, etc., on the rate of an enzyme-catalyzed reaction. In addition, enzymes can be applied as histochemical reagents to remove macromolecular substrates, examples being RNases, DNases, amylases, etc., or to identify substrates after their fluorescent labeling. Chromo- and fluorogenic reactions are usually employed to reveal enzymatic activity. A relevant issue to avoid diffusion artifacts or false localization is that the colored or fluorescent product of a given enzymatic reaction must have very scarce or null water solubility. However, insoluble or crystalline precipitates should be not confused with specific cell structures. Some examples will illustrate these points.

Keywords: AEC, Arylsulfatases, Benzothiazole-hydroxystyrene, CARD, Caspases, Cathepsins, Dehydrogenases, ELF, Esterases, Fluorogenic substrates, Glycosidases, Indigogenic methods, Peroxidases, Phosphatases, Tyramide signal amplification.

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