In Situ Zymography

The techniques of Western blotting and immunocytochemistry are suitable for the quantification and localization, respectively, of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) expression. However, they do not indicate the endogenous balance of matrix degrading activity and inhibition because: 1) at present very few antibodies distinguish between the precursor and proteolytically processed forms of MMPs; and 2) TIMPs present in the tissue sample can prevent matrix degradation by MMPs even if the enzymes are in the active form. Although biochemical studies can determine net MMP activity they do not provide any localizational information and activation of MMPs may occur during disruption of the tissue sample. In situ zymography not only enables the estimation of net MMP activity but also allows the localization of this activity in tissue sections. This technique was previously used for detection of enzymatic activity released by explants of developing amphibian tissue (1 ), allows preservation of the histology and does not require species-specific reagents (2 ). It has been recently used to detect MMP activity in human atherosclerotic plaques (3 ,4 ) and saphenous vein organ cultures (5 ,6 ).