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In Situ Zymography and Immunolabeling in Fixed and Decalcified Craniofacial Tissues

Isabel M. Porto, Lenaldo B. Rocha, Marcos A. Rossi and Raquel F. Gerlach

Department of Morphology, Dental School of Piracicaba, University of Campinas, Piracicaba, São Paulo, Brazil (IMP), and Department of Cellular and Molecular Biology and Pathogenic Bioagents (LBR) and Department of Pathology (MAR), Medicine School of Ribeirão Preto, and Department of Morphology, Stomatology and Physiology, Dental School of Ribeirão Preto (RFG), University of São Paulo, Ribeirão Preto, São Paulo, Brazil

Correspondence to: Dr. Raquel F. Gerlach, Departamento de Morfologia, Estomatologia e Fisiologia, Faculdade de Odontologia de Ribeirão Preto, FORP/USP, Avenida do Café, S/N CEP 14040-904 Ribeirão Preto, SP, Brazil. E-mail: rfgerlach{at}forp.usp.br

In situ zymography is a very important technique that shows the proteolytic activity in sections and allows researchers to observe the specific sites of proteolysis in tissues or cells. It is normally performed in non-fixed frozen sections and is not routinely performed in calcified tissues. In this study, we describe a technique that maintains proteolytic activity in fixed and decalcified sections obtained after routine paraffin sectioning in conventional microtome and cryostat sections. We used adult rat hemimandibles, which presented bone, enamel, and dentine matrices; the substrate used was dye-quenched-gelatin. Gelatinolytic activity was colocalized with MMP-2 using fluorescent antibodies. Specific proteolytic activity was observed in all sections, compatible with metalloproteinase activity, particularly in dentine and bone. Furthermore, matrix metalloproteinase-2 was colocalized to the sites of green fluorescence in dentine. In conclusion, the technique presented here will allow in situ zymography reactions in fixed, decalcified, and paraffin-embedded tissues, and we showed that paraformaldehyde-lysine-periodate–fixed cryostat sections are suitable for colocalization of gelatinolytic activity and protein labeling with antibodies. (J Histochem Cytochem 57:615–622, 2009)

Key Words: craniofacial tissues • in situ zymography • dye-quenched-gelatin • proteolytic activity • fluorescence microscopy • immunolabeling • matrix metalloproteinase-2 • dentine • bone

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