In Situ Zymography and Immunolabeling in Fixed and Decalcified Craniofacial Tissues

Isabel M. Porto ¹, Lenaldo B. Rocha ¹, Marcos A. Rossi ¹ and Raquel F. Gerlach ^1*

¹ Department of Morphology, Dental School of Piracicaba, University of Campinas, FOP/UNICAMP, Piracicaba, SP, Brazil (IMP); Department of Cellular and Molecular Biology and Pathogenic Bioagents (LBR) and Department of Pathology (MAR), Medicine School of Ribeirão Preto, University of São Paulo, FMRP/USP, Ribeirão Preto, SP, Brazil; and Department of Morphology, Stomatology and Physiology, Dental School of Ribeirão Preto, University of São Paulo, FORP/USP, Ribeirão Preto, SP, Brazil (RFG)

^* To whom correspondence should be addressed. E-mail: rfgerlach{at}forp.usp.br.

Submitted on June 20, 2008
Accepted on 22 January 2009

	Abstract

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 yet. In the present study, we describe a technique that maintains proteolytic activity in fixed and decalcified sections obtained after routine paraffin sectioning in a conventional microtome and in cryostat sections. We used adult rat hemimandibles, which presented bone, enamel and dentine matrices, and the substrate used was DQ-gelatin. Gelatinolytic activity was co-localized with MMP-2 using fluorescent antibodies. Specific proteolytic activity was observed in all sections, compatible with metalloproteinase activity, particularly in dentine and bone. Furthermore, MMP-2 was co-localized 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 PLP-fixed cryostat sections are suitable for co-localization of gelatinolytic activity and protein labeling with antibodies.

Key Words: Craniofacial tissues, in situ zymography, DQ-gelatin, proteolytic activity, fluorescence microscopy, immunolabeling, matrix metalloproteinase-2, dentine, bone

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