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.