Protocol

Matrix Metalloproteinase Protocols

Volume 622 of the series Methods in Molecular Biology pp 271-277

Date:

In Situ Zymography

  • Sarah J. GeorgeAffiliated withBristol Heart Institute, Bristol Royal Infirmary, University of Bristol
  • , Jason L. JohnsonAffiliated withBristol Heart Institute, Bristol Royal Infirmary, University of Bristol

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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.

Key words

Matrix-degrading metalloproteinases in situ localisation proteolytic activity