Precise localization of gelatinase activity in sections and cells became possible with the introduction of dye-quenched (DQ)-gelatin, which is gelatin that is heavily labeled with FITC molecules so that its fluorescence is quenched (Oh et al. 1999; Curry et al. 2001; Duchossoy et al. 2001; Goodall et al. 2001; Lindsey et al. 2001; Teesalu et al. 2001; Wang and Lakatta 2002; Zhang and Salamonsen 2002; Mook et al. 2003; Platt et al. 2003; Lee et al. 2004). After cleavage of DQ-gelatin by gelatinolytic activity, fluorescent peptides are produced that can be visualized against a weakly fluorescent background (EnzCheck; Molecular Probes, Eugene, OR). The substrate was developed for assaying protease activity in solutions, but the substrate has properties that enable localization of protease activity in cells or tissues under certain conditions. Oh et al. (1999) incubated unfixed cryostat sections of developing optic nerves and cultured live oligodendrocytes with an aqueous medium containing DQ-gelatin. After overnight incubation without any further fixation or washes, gelatinolytic activity was localized and photographed. This procedure enabled localization of MMP-2 and MMP-9 activity in developing optic nerves. It appeared that the activity had a similar distribution pattern as myelin basic protein. Activity was also found pericellularly at growing tips of oligodendrocytes. Formation of fluorescent peptides was prevented by addition of phenanthroline or TIMP-1 to the incubation media, which indicates that MMP activity was demonstrated. Parallel investigations on MMP-2 and MMP-9 of oligodendrocytes with zymography led to the conclusion that MMP-9 was the gelatinase involved in myelin formation by oligodendrocytes. The same procedure was recently applied by Lee et al. (2004), who localized gelatinolytic activity due to MMP-9 in pyramidal and granular neurons of the hippocampus after ischemia. An important role for MMP-9 in the pathogenesis of neuronal damage was proposed. Curry et al. (2001) added 1% agarose to DQ-gelatin in analogy with the principle introduced by Galis et al. (1995). After spreading this solution on glass slides and gelling at 4C, unfixed cryostat sections were mounted on the gelatin substrate, coverslipped, and incubated for 20 hr at 37C. Fluorescence was detected in rat ovaries during follicular growth, ovulation, and early luteal formation. Production of fluorescence was prevented by EDTA and ilomastat, a synthetic MMP inhibitor. Therefore, it was concluded that gelatinolytic activity by MMPs was demonstrated in these tissues. Teesalu et al. (2001) used DQ-gelatin, agar, and unfixed cryostat sections to localize gelatinolytic activity in inflammatory lesions caused by experimental autoimmune encephalomyelitis and demonstrated that MMP-9 was responsible for the in situ gelatin breakdown on the basis of parallel zymography experiments.
MMP-9 activity was detected in the vicinity of infiltrating neutrophils in canine myocardium subjected to ischemia and reperfusion with the use of DQ-gelatin applied to unfixed cryostat sections (Lindsey et al. 2001). Gelatinolytic activity was inhibited by both EDTA and neutralizing MMP-9 antibody. Moreover, gelatinolytic activity was not observed in non-ischemic myocardium. It was concluded that infiltrating neutrophils were the source of active MMP-9.
MMP-2 activity was localized on elastin fibers in blood vessel walls after incubation of cryostat sections of inferior mesenteric veins of patients with abdominal aortic aneurysms with DQ-gelatin (Goodall et al. 2001). Phenanthroline inhibited production of fluorescence and gelatin zymography demonstrated the presence of active MMP-2. The authors concluded that MMP-2 plays a primary role in aneurysm formation. However, it should be noted that autofluorescence of elastin fibers may interfere with detection of fluorescence due to gelatinolytic activity.
Duchossoy et al. (2001) combined in situ zymography using DQ-gelatin with immunodetection of laminin to study the role of MMPs in spinal cord injury. For this purpose, unfixed spinal cord cryostat sections were incubated overnight in medium containing DQ-gelatin and antibody against laminin. After a rapid wash, sections were incubated with secondary antibodies and studied with fluorescence microscopy. Gelatinolytic activity was co-localized with laminin surrounding blood vessels in injured spinal cord and was detected in lesion sites in and around infiltrating cells. Preincubation with blocking antibodies against MMP-2 and MMP-9 strongly reduced fluorescence intensity, suggesting the involvement of gelatinase activity. The authors concluded that gelatinases play a role in blood–spinal barrier disruption, leukocyte infiltration, disruption of the ECM, and clearance of debris after spinal cord injury. Because it is difficult to imagine that soluble fluorescent peptides produced by cleavage of DQ-gelatin can be detected after a wash of the sections and additional incubation with secondary antibodies, it must be concluded that extracellular localization of gelatinase activity must be the result of binding of fluorescent peptides to MMPs in the ECM.
Zhang and Salamonsen (2002) were not successful in combining in situ zymography using DQ-gelatin and immunostaining of MMPs on the same section of human endometrium. This may be due to the fact that cryostat sections were fixed in formalin and 2% gelatin was added to the DQ-gelatin-containing incubation medium. It has been established that fixation with formaldehyde and therefore no doubt with formalin, completely inhibits gelatinolytic activity (Mook et al. 2003). Moreover, the addition of 2% gelatin to 0.01% DQ-gelatin may reduce the efficacy of breakdown of DQ-gelatin. In situ zymography without simultaneous IHC detection of MMP-2 and MMP-9 resulted in findings on the basis of which it was concluded that MMPs were more active in menstrual endometrium compared with other stages of the cycle, which suggests that MMPs play a role in matrix degradation during menstruation. Wang and Lakatta (2002) demonstrated gelatinolytic activity to be increased in the wall of rat aortas in relation with aging using DQ-gelatin. However, detailed descriptions of the method they applied were lacking. It was concluded that activity mainly resulted from MMP-2 because activity was almost completely inhibited by a blocking antibody against MMP-2. The role of MMP-2 and MMP-9 in rat sciatic nerves after crush and during regeneration was established with in situ zymography with DQ-gelatin and gel zymography (Platt et al. 2003). The fluorescence due to gelatinase activity at the lesion site was prevented by addition of phenanthroline.
The first application to cancer of in situ zymography with DQ-gelatin was performed by Mook et al. (2003). The procedure was based on the incubation of unfixed cryostat sections of rat liver containing colon cancer metastases using a low-gelling temperature (LGT) agar- and DQ-gelatin-containing incubation medium. The time of incubation was only 60 min, the method could be combined with IHC on the same section, and the method was extensively tested for its specificity. Gelatinolytic activity due to MMP-2 was detected in stroma of colon cancer metastases (Figure 4) .
It can be concluded that the use of DQ-gelatin instead of labeled or unlabeled gelatin is superior for in situ zymography because fluorescence is produced at sites of gelatinolytic activity instead of decreased staining intensity at gelatinolytic areas. However, the limitations described above for in situ gelatin zymography apply for DQ-gelatin as well. Moreover, autofluorescence of the tissue should be carefully inspected by incubation of tissue sections with incubation media that lack the substrate.