In Situ Zymography: A Molecular Pathology Technique to Localize Endogenous Protease Activity in Tissue Sections

Yan, Shaohua; Blomme, Eric A.G.

doi:10.1354/vp.40-3-227

Cited by 55 publications

(49 citation statements)

References 51 publications

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“…Intracellular and extracellular cleavage of collagen type IV was detected that was largely due to intracellular and extracellular cathepsin B activities. Yan and Blomme (2003) demonstrated breakdown of DQ-collagen type IV in cryostat sections of rat tibia. Fluorescence was observed in the distal portion of the hypertrophic zone of growth plates of the tibia.…”

Section: In Situ Zymography With Other Quenched Fluorogenic Substratesmentioning

confidence: 91%

Metabolic Mapping of Proteinase Activity with Emphasis on In Situ Zymography of Gelatinases

Frederiks

Mook

2004

J Histochem Cytochem.

111

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S U M M A R Y Proteases are essential for protein catabolism, regulation of a wide range of biological processes, and in the pathogenesis of many diseases. Several techniques are available to localize activity of proteases in tissue sections or cell preparations. For localization of the activity of matrix metalloproteinases, in situ zymography was introduced some decades ago. The procedure is based on zymography using SDS polyacrylamide gels containing gelatin, casein, or fibrin as substrate. For in situ zymography, either a photographic emulsion containing gelatin or a fluorescence-labeled proteinaceous macromolecular substrate is brought into contact with a tissue section or cell preparation. After incubation, enzymatic activity is revealed as white spots in a dark background or as black spots in a fluorescent background. However, this approach does not allow precise localization of proteinase activity because of limited sensitivity. A major improvement in sensitivity was achieved with the introduction of dye-quenched (DQ-)gelatin, which is gelatin that is heavily labeled with FITC molecules so that its fluorescence is quenched. After cleavage of DQ-gelatin by gelatinolytic activity, fluorescent peptides are produced that are visible against a weakly fluorescent background. The incubation with DQ-gelatin can be combined with simultaneous immunohistochemical detection of a protein on the same section. To draw valid conclusions from the findings with in situ zymography, specific inhibitors need to be used and the technique has to be combined with immunohistochemistry and zymography. In that case, in situ zymography provides data that extend our understanding of the role of specific proteinases in various physiological and pathological conditions.

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Section: In Situ Zymography With Other Quenched Fluorogenic Substratesmentioning

confidence: 91%

Metabolic Mapping of Proteinase Activity with Emphasis on In Situ Zymography of Gelatinases

Frederiks

Mook

2004

J Histochem Cytochem.

111

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“…16 Cryosections were air-dried for 5 minutes in room temperature and washed with phosphate-buffered saline. Sections were incubated for 2 hours in 37°C covered with DQ-gelatin (0.1 g/L in reaction buffer, EnzChek gelatinase/collagenase assay kit, Invitrogen).…”

Section: In Situ Zymographymentioning

confidence: 99%

Cerebral Mast Cells Mediate Blood-Brain Barrier Disruption in Acute Experimental Ischemic Stroke Through Perivascular Gelatinase Activation

Mattila

Strbian

Saksi

et al. 2011

Stroke

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Background and Purpose-Perivascularly positioned cerebral mast cells (MC) have been shown to participate in acute blood-brain barrier disruption and expansive brain edema following experimental transient cerebral ischemia. However, the underlying molecular mechanisms remain unknown. Because proteolytic gelatinase enzymes, matrix metalloproteinases (MMP)-2 and MMP-9, are thought to have a central role in compromising the integrity of the blood-brain barrier following ischemia, we examined whether cerebral MCs influence gelatinase activity in ischemic cerebral microvasculature. Methods-Rats underwent 60 minutes of middle cerebral artery occlusion followed by 3-hour reperfusion, and were treated with a MC-stabilizing (cromoglycate), or Key Words: mast cells Ⅲ gelatinases Ⅲ blood-brain barrier E xpansive brain edema is a frequent cause of death after large ischemic strokes, 1 as it reduces blood flow in the ischemic penumbra and can lead to herniation of brain structures. Edema is predominantly caused by functional and structural disturbance of the blood-brain barrier (BBB). Proteolytic gelatinase enzymes, most importantly matrix metalloproteinases (MMP)-2 and MMP-9, are considered to be central mediators of ischemic BBB disruption; this is because of their ability to degrade components of microvascular basal lamina, especially collagen type IV, 2-4 and to disrupt tight junction proteins. 5 The mechanisms triggering early activation of gelatinases in ischemic brain remain under active debate. 2,6 Mast cells (MC) are resident inflammatory cells positioned in the outer layers of various tissues (eg, mucosa, epithelia, and vasculature) in mammals, including the central nervous system. 7 We and others have previously shown that MCs have a role in the pathophysiology of ischemic, hypoxic-ischemic, and hemorrhagic brain injury. 8 -11 Both pharmacological inhibition and genetic deficiency of MCs led to a significant reduction in postischemic BBB disruption, cerebral edema, and neutrophil infiltration, 8 presenting MC inhibition as a potential therapeutic avenue to prevent inflammatory damage to the neurovasculature, especially in conjunction with thrombolytics. 9 We envisaged a molecular pathway for ischemic BBB disruption where factors liberated from MCs might fuel gelatinase activation within the targeted neurovascular unit and lead to degradation of the microvascular wall, especially Materials and Methods AnimalsThe in vivo methodology has been described in detail previously. 8 Briefly, adult, male, Wistar rats (Harlan Nederland) and WsRc Ws/Ws rats (Japan SLC, Inc), 290 to 340g, were anesthetized with ketamin hydrochloride (intraperitoneal, 50 mg/kg, Ketalar, Parke-Davis) and medetomidine hydrochloride (subcutaneous, 0.5 mg/kg, Domitor, Orion). The left femoral artery and vein were cannulated for measurements of blood pressure, arterial pH, blood gases, and blood glucose and drug and/or vehicle infusions. Cerebral blood flow was measured on-line with laser-Doppler flowmetry (Oxy-Flow, Oxford Optronix) as described...

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“…20 -22 Recently, a new technique has been introduced to measure gelatinolytic activity in histological cross sections with a high spatial resolution. 23,24 We have adopted this method for vascular tissue and incorporated it into 3-dimensional histological reconstructions. Our second aim was to evaluate, using a combination of the above techniques, whether MMPs are active very locally or multiple cell types are involved in experimental atherosclerosis in vivo.…”

Section: Clinical Perspective P 616mentioning

confidence: 99%

Gelatinolytic Activity in Atherosclerotic Plaques Is Highly Localized and Is Associated With Both Macrophages and Smooth Muscle Cells In Vivo

et al. 2007

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Background-Atherosclerosis is considered an inflammatory disease. Recent studies provided evidence for a predominant upstream location of plaque inflammation. The present study introduces a novel technique that evaluates the underlying mechanism of this spatial organization. Methods and Results-In hypercholesterolemic rabbits, atherosclerosis of the infrarenal aorta was induced by a combination of endothelial denudation and a high-cholesterol diet (2% cholesterol for 2 months). At the time of death, aortic vessel segments were dissected and reconstructed with a new technique that preserved the original intravascular ultrasound-derived lumen geometry. This enabled us to study the spatial relation of histological markers like macrophages, smooth muscle cells, lipids, gelatinolytic activity, and oxidized low-density lipoprotein. Results showed a predominant upstream localization of macrophages and gelatinase activity. Colocalization studies indicated that gelatinase activity was associated with macrophages and smooth muscle cells. Further analysis revealed that this was caused by subsets of smooth muscle cells and macrophages, which were associated with oxidized low-density lipoprotein accumulation. Conclusions-Upstream

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In Situ Zymography: A Molecular Pathology Technique to Localize Endogenous Protease Activity in Tissue Sections

Cited by 55 publications

References 51 publications

Metabolic Mapping of Proteinase Activity with Emphasis on In Situ Zymography of Gelatinases

Metabolic Mapping of Proteinase Activity with Emphasis on In Situ Zymography of Gelatinases

Cerebral Mast Cells Mediate Blood-Brain Barrier Disruption in Acute Experimental Ischemic Stroke Through Perivascular Gelatinase Activation

Gelatinolytic Activity in Atherosclerotic Plaques Is Highly Localized and Is Associated With Both Macrophages and Smooth Muscle Cells In Vivo

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