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FASEB J. 1995 Jul;9(10):974-80.

Microscopic localization of active proteases by in situ zymography: detection of matrix metalloproteinase activity in vascular tissue.

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Vascular Medicine and Atherosclerosis Unit, Brigham and Women's Hospital, Boston, Massachusetts, USA.


Many physiological and pathological processes involve tissue remodeling due in part to degradation of extracellular matrix. Several factors limit current approaches used for detection of matrix-degrading enzymes in tissues. Matrix metalloproteinases (MMPs), enzymes specialized in catabolism of extracellular matrix constituents, require processing from inactive zymogen precursors to gain enzymatic function. Presently available antibodies do not distinguish between precursor and proteolytically processed forms of MMPs. Also, ubiquitous tissue inhibitors of metalloproteinases (TIMPs) could prevent matrix degradation by MMPs even if the enzymes were in an active form. For these reasons immunocytochemistry does not provide information regarding the functional state of these enzymes. Biochemical studies of tissue extracts preclude localization and entail the possibility of artifactual activation of the enzymes consequent to tissue disruption. To obviate these problems, we have adapted substrate zymography to frozen tissue sections to assess net proteolytic activity in situ. We report here the details and the validation of this methodology. Initial experiments defined casein fluorescently labeled with resorufin as a useful substrate for detecting stromelysin, and fluoresceinated gelatin or autoradiographic emulsion as suitable for detecting gelatinolytic activity by this approach. Either TIMP-1 or the Zn chelator 1,10-phenanthroline reduced the zymographic activity in cryosections of atheroma from humans or rabbits. Inhibitors of serine proteases did not reduce the extent of substrate lysis substantially. In situ zymography preserves the fine morphological details of the tissue and can complement the study of enzyme expression by other microscopic techniques, such as immunocytochemistry. This approach may prove generally applicable for the detection of protease activity in tissue sections permitting exploration of the roles of these enzymes in pathobiology.

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