In Situ Zymography and Immunolabeling in Fixed and Decalcified Craniofacial Tissues
AUTOR(ES)
Porto, Isabel M.
FONTE
Histochemical Society
RESUMO
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)
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2699317Documentos Relacionados
- Use of Discriminatory Probes for Strain Typing of Formalin-Fixed, Rabies Virus-Infected Tissues by In Situ Hybridization
- Differentiation of foetal and maternal erythrocytes in formol-fixed tissues.
- Microtubule distribution in cultured cells and intact tissues: improved immunolabeling resolution through the use of reversible embedment cytochemistry.
- PCR in situ hybridisation detection of HPV 16 in fixed CaSki and fixed SiHa cell lines.
- Bone induction in implants of decalcified bone and dentine.