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Gelatinase in situ zymography

Posted on Thursday, January 18, 2007

Description
DQ-gelatin (dye-quenched) is heavily labeled with FITC molecules and its fluorescence is quenched. Gelatinolysis produces fluorescent peptides. This assay allows the study of the spatial distribution of gelatinolytic activity in frozen tissue sections or live cell cultures.

Procedure
1- Dissect the specimen and rinse in cold PBS. Do not fixate.
2- Immerse the specimen in embedding medium for frozen tissue (poly-ethylene-glycol based).
3- Freeze quickly on dry ice or liquid nitrogen.
4- Slice in 15 um thick cryostat sections and mount on albumin coated (not gelatin coated) glass slides. Samples can be stored at -70 degree C.
5- To perform the assay, thaw the samples and incubate with reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, pH 7.6) containing 30 ug/ml of DQ-gelatin (Molecular Probes) for 2 hs at 37 degree C.
6- Wash very gently with the same buffer to avoid displacement of fluorescent peptides.
7- Mount in aqueous mounting medium and examine under fluorescent microscope.


Recipes


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Tips
To validate the results, use specific inhibitors and perform immunohistochemistry in adjacent slices. However, keep in mind that this assay detects active gelatinases, then pro-gelatinases may not be detected even if they were labeled with antibodies.
It is not necessary to freeze cell cultures but incubation time should be assessed for each cell type and culture density.


Submitted by: Ana Laura

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