Incorporation of fluorescent enzyme substrates in agarose gel for in situ zymography

The currently available methods for the detection of proteases in tissue se ctions are characterized by limited substrate specificity and low sensitivi ty and are also cumbersome. We have developed a novel in situ zymography me thod that uses a synthetic substrate conjugated to a fluorescent tag for de tection of proteases in tissue sections. In the presence of active enzyme, the fluorescent tag is cleaved off from the substrate peptide chain resulti ng in an approximately 100-fold increase in the fluorescent signal. In orde r to minimize the diffusion of the fluorescent tag, the substrate is incorp orated into 1% agarose prior to overlaying onto the tissue section. This me thod retains the morphological details of the tissue section, is highly sen sitive and specific for the designated peptide sequence, and provides infor mation regarding the functional status of the enzyme. Thus, this method cou ld be used for detection and monitoring of enzymatic activity in tissue sec tions for a variety of applications. (C) 2001 Academic Press.