In Situ Zymography and Localization of Bright Green Fluorescent Gelatinase Activity in Tissue Sections

Abstract

In situ zymography is an efficient and low-cost technique to detect the activity of the gelatinases matrix metalloproteinase (MMP)-2 and MMP-9 in various tissues from humans and experimental animal models. This technique also shows the precise localization of gelatinases in large or small tissues and in cells. It is a one-day technique that uses a dye-quenched fluorescent gelatin (Dq-gelatin). The Dq-gelatin is loaded into tissue sections and upon its interaction with local active gelatinases, it is proteolyzed into highly fluorescent peptides that emit bright green fluorescence, thus reflecting the level of activity and localization of the gelatinases. Combined with other biochemical techniques in tissue homogenates and fractions, the in situ zymography provides a fluorescence microscopy and digital imaging technique to assess the subcellular localization and the role of gelatinases in tissue remodeling in health and disease.

Keywords: Extracellular matrix; Gelatin; Matrix metalloproteinases; Remodeling; Vascular.

Fig. 1.

Detection of increased gelatinase activity in aortas of animal model of hypertension using in situ zymography. Aortas from Sham-operated and two kidney, one clip (2K-1C) hypertensive Sprague Dawley rats were cut into 0.5 μm cryosections, loaded with Dq-gelatin, and examined under fluoresce microscope total magnification 400. The bright green fluorescence of the MMP gelatinase activity in the aortic intima, media and adventitia layers was visualized, and snap-pictures were captured (A). Negative control experiments were performed in parallel on aortas from Sham rat without Dq gelatin. All tissue sections were counter-stained with DAPI to detect the cellular nuclei (B). Bright green fluoresce of gelatinase activity was quantified using ImageJ, and the cumulative data from different aortic tissue sections were analyzed using Graph-Pad Prism software (C). L= lumen, M = media, Adv = adventitia, horizontal calibration bar = 20 μM. Data represent means±SEM of aortic tissue sections from 5 rats. * Intensity of bright green fluorescence is significantly higher in aortas from 2K-1C hypertensive rats vs Sham rats (p<0.05).