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Journal of Histochemistry and Cytochemistry, Vol. 45, 779-784, Copyright © 1997 by The Histochemical Society, Inc.

RAPID COMMUNICATION

Use of Enzyme Overlay Membranes to Survey Proteinase Activity in Frozen Sections: Cathepsin-like and Plasmin-like Activity in Regenerating Newt Limbs

Frances A. Daya and Daniel A. Neufelda
a University of South Dakota School of Medicine, Department of Anatomy and Structural Biology, Vermillion, South Dakota

Correspondence to: Daniel A. Neufeld, U. of South Dakota School of Medicine, Dept. of Anatomy and Structural Biology, Vermillion, SD 57069.

We present a method that permits extremely simple and rapid screening of proteolytic enzyme activity in sectioned tissues. Enzyme overlay membranes (EOMs) are custom-made membranes designed to fluoresce at sites of specific proteolytic enzyme activity after separation of proteins by gel electrophoresis. EOMs, selected to detect either plasmin-like or cathepsin B-like activity, have been used in a novel way to document the distribution of enzyme activity in frozen sectioned tissues. When moistened membranes were placed in contact with sectioned regenerating newt limbs, a fluorescent pattern of enzyme activity was generated. In limbs at 3 hr post amputation, cathepsin B-like activity was prominent across the amputation site but plasmin-like activity was distributed in dermal and deeper proximal tissues, suggesting different roles for these two classes of enzymes. EOM enzymology in situ (EEI) on frozen sectioned tissues may be a widely useful technique to display distribution and level of activity of proteolytic enzymes in various systems. (J Histochem Cytochem 45:779-783, 1997)

Key Words: enzyme overlay membranes, proteolytic enzymes, protein separation, newt, cathepsin B, plasmin


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