Fluorescence histochemical detection of hydrolases in tissue sections and culture cells

Abstract

Fluorescence and dye histochemical methods are compared for the investigation of hydrolases in sections and culture cells. At present, only some of the synthetic substrates with fluorescent leaving groups may be used for the fluorescence localization of these enzymes in sections. This limitation is due to a reduced fluorescence intensity and/or diffusion of the fluorescent tags. Satisfactory results are obtained for alkaline phosphatase, non-specific esterases and proteases with naphthol AS and 4-methoxy-2-naphthylamine coupled to nitrosalicylaldehyde. If, however, cultured monolayer cells are investigated, all synthetic substrates with fluorescent tags are suitable, including those that have so far only been used for biochemical hydrolase measurements. The fluorescent leaving groups are naphthol AS and its derivates, 4-methoxy-2-naphthylamine, aminomethylcoumarin, aminomethyltrifluoromethylcoumarin, methylumbelliferon, fluorescein and, with some limitations, also 1- and 2-naphthol. These fluorescence methods are more sensitive than the corresponding dye procedures. In addition, the fluorescence techniques allow the use of more synthetic substrates and therefore more information become available than with dye histochemistry about the enzymic properties of culture cells.