Abstract
In situ zymography is a unique laboratory technique that enables the localisation of matrix-degrading metalloproteinase (MMP) activity in histological sections. Frozen sections are placed on glass slides coated with fluorescently labelled matrix proteins. After incubation MMP activity can be observed as black holes in the fluorescent background due to proteolysis of the matrix protein. Alternatively frozen sections can be incubated with matrix proteins conjugated to quenched fluorescein. Proteolysis of the substrate by MMPs leads to the release of fluorescence. This technique can be combined with immunohistochemistry to enable co-location of proteins such as cell type markers or other proteins of interest. Additionally, this technique can be adapted for use with cell cultures, permitting precise location of MMP activity within cells, time-lapse analysis of MMP activity and analysis of MMP activity in migrating cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Gross, J. and Lapiere, C. M. (1962) Collagenolytic activity in amphibian tissues: a tissue culture assay. Proc Natl Acad Sci U S A 48, 1014–1022.
Sappino, A. P., Huarte, J., Vassalli, J. D., and Belin, D. (1991) Sites of synthesis of urokinase and tissue-type plasminogen activators in the murine kidney. J Clin Invest 87, 962–970.
Galis, Z. S., Muszynski, M., Sukhova, G. K., Simon-Morissey, E., Unemori, E. N., Lark, M. W., Amento, E., and Libby, P. (1994) Cytokine-stimulated human vascular smooth muscle synthesize a complement of enzymes required for extracellular matrix digestion. Circ Res 75, 181–189.
George, S. J., Baker, A. H., Angelini, G. D., and Newby, A. C. (1998) Gene transfer of tissue inhibitor of metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in human saphenous veins. Gene Ther 5, 1552–1560.
George, S. J., Johnson, J. L., Angelini, G. D., Newby, A. C., and Baker, A. H. (1998) Adenovirus-mediated gene transfer of the human TIMP-1 gene inhibits SMC migration and neointima formation in human saphenous vein. Hum Gene Ther 9, 867–877.
Johnson, J. L., Jackson, C. L., Angelini, G. D., and George, S. J. (1998) Activation of matrix-degrading metalloproteinases by mast cell proteases in atherosclerotic plaques. Arterioscler Thromb Vasc Biol 18, 1707–1715.
Oh, L., Larsen, P., Krekoski, C., Edwards, D., Donovan, F., Werb, Z., and Yong, V. (1999) Matrix metalloproteinase-9/gelatinase B is required for process outgrowth by oligodendrocytes. J Neurosci 19, 8464–8475.
Magnoni, S., Baker, A., George, S. J., Duncan, W. C., Kerr, L. E., McCulloch, J., and Horsburgh, K. (2004) Differential alterations in the expression and activity of matrix metalloproteinases (MMPs) 2 and 9 after transient cerebral ischemia in mice. Neurobiol Dis 17, 188–197.
Magnoni, S., Baker, A., Thomson, S., Jordan, G., George, S. J., McColl, B. W., McCulloch, J., and Horsburgh, K. (2007) Neuroprotective effect of adenoviral-mediated gene transfer of TIMP-1 and-2 in ischemic brain injury. Gene Therapy 14, 621–625.
Galis, Z. S., Sukhova, G. K., and Libby, P. (1995) Microscopic localization of active proteases by in situ zymography: detection of matrix metalloproteinase activity in vascular tissue. FASEB J 9, 974–980.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
George, S.J., Johnson, J.L. (2010). In Situ Zymography. In: Clark, I. (eds) Matrix Metalloproteinase Protocols. Methods in Molecular Biology, vol 622. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-299-5_17
Download citation
DOI: https://doi.org/10.1007/978-1-60327-299-5_17
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60327-298-8
Online ISBN: 978-1-60327-299-5
eBook Packages: Springer Protocols