Limits...
A method for rapid demineralization of teeth and bones.

Cho A, Suzuki S, Hatakeyama J, Haruyama N, Kulkarni AB - Open Dent J (2010)

Bottom Line: The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate.This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest.However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase.

View Article: PubMed Central - PubMed

Affiliation: Gene Targeting Facility, Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Bethesda, MD 20892, USA.

ABSTRACT
Tooth and bone specimen require extensive demineralization for careful analysis of cell morphology, as well as gene and protein expression levels. The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate. This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest. To analyze LacZ activity, and the expression of other genes and their protein products in teeth and bones, it is necessary to carry out a complete demineralization of the specimen before cutting sections. However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase. Therefore, most protocols currently use mild acids such as 0.1 M ethylene diamine tetra-acetic acid (EDTA) for demineralization of tooth and bone specimen, which require a longer period of treatment for complete demineralization. A method by which hard tissue specimens such as teeth and bones can be rapidly, but gently, decalcified is necessary to save time and effort. Here, we report a suitable method for rapid demineralization of mouse teeth in 0.1M EDTA at 42˚C without any loss of ß-galactosidase activity.

No MeSH data available.


Related in: MedlinePlus

The choice of the decalcifier reagent affects β-galactosidase activity. The 3-month-old Dspp-lacZ mouse skulls were fixed with 4% PFA and decalcified with either Formical-4 (a), Immunocal (b), or 0.1M EDTA in PBS (c). Absence of blue staining in the odontoblasts of sections (a) and (b) indicates a lack of β-galactosidase enzyme activity. Only the incisor sections from the skulls decalcified with the EDTA- based demineralization agent preserved the activity, as indicated by the blue product of the β-galactosidase substrate (c).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3040998&req=5

Figure 3: The choice of the decalcifier reagent affects β-galactosidase activity. The 3-month-old Dspp-lacZ mouse skulls were fixed with 4% PFA and decalcified with either Formical-4 (a), Immunocal (b), or 0.1M EDTA in PBS (c). Absence of blue staining in the odontoblasts of sections (a) and (b) indicates a lack of β-galactosidase enzyme activity. Only the incisor sections from the skulls decalcified with the EDTA- based demineralization agent preserved the activity, as indicated by the blue product of the β-galactosidase substrate (c).

Mentions: EDTA based demineralization maintains β-galactosidase activity: The acidity of demineralization agents has been reported to affect the stability of proteins, enzyme activities and tissue integrity [8]. Therefore, we analyzed the effects of various demineralization reagents on the β-galactosidaseactivity. We fixed the skulls from 3-month-old Dspp-LacZ mice with 4% PFA and then decalcified them in Formical-4 and Immunocal solution, both of which are formic acid-based fixative decalcifiers. We then prepared the frozen sections, stained them with LacZ solution and compared the β-galactosidase activity with the sections from the skulls that were fixed by 4% PFA and decalcified with 0.1M EDTA. Only the tooth sections from the skulls treated with EDTA-based decalcifiers preserved the enzyme activity, as shown by the strong blue staining in the odontoblasts in Fig. (3c).


A method for rapid demineralization of teeth and bones.

Cho A, Suzuki S, Hatakeyama J, Haruyama N, Kulkarni AB - Open Dent J (2010)

The choice of the decalcifier reagent affects β-galactosidase activity. The 3-month-old Dspp-lacZ mouse skulls were fixed with 4% PFA and decalcified with either Formical-4 (a), Immunocal (b), or 0.1M EDTA in PBS (c). Absence of blue staining in the odontoblasts of sections (a) and (b) indicates a lack of β-galactosidase enzyme activity. Only the incisor sections from the skulls decalcified with the EDTA- based demineralization agent preserved the activity, as indicated by the blue product of the β-galactosidase substrate (c).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040998&req=5

Figure 3: The choice of the decalcifier reagent affects β-galactosidase activity. The 3-month-old Dspp-lacZ mouse skulls were fixed with 4% PFA and decalcified with either Formical-4 (a), Immunocal (b), or 0.1M EDTA in PBS (c). Absence of blue staining in the odontoblasts of sections (a) and (b) indicates a lack of β-galactosidase enzyme activity. Only the incisor sections from the skulls decalcified with the EDTA- based demineralization agent preserved the activity, as indicated by the blue product of the β-galactosidase substrate (c).
Mentions: EDTA based demineralization maintains β-galactosidase activity: The acidity of demineralization agents has been reported to affect the stability of proteins, enzyme activities and tissue integrity [8]. Therefore, we analyzed the effects of various demineralization reagents on the β-galactosidaseactivity. We fixed the skulls from 3-month-old Dspp-LacZ mice with 4% PFA and then decalcified them in Formical-4 and Immunocal solution, both of which are formic acid-based fixative decalcifiers. We then prepared the frozen sections, stained them with LacZ solution and compared the β-galactosidase activity with the sections from the skulls that were fixed by 4% PFA and decalcified with 0.1M EDTA. Only the tooth sections from the skulls treated with EDTA-based decalcifiers preserved the enzyme activity, as shown by the strong blue staining in the odontoblasts in Fig. (3c).

Bottom Line: The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate.This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest.However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase.

View Article: PubMed Central - PubMed

Affiliation: Gene Targeting Facility, Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Bethesda, MD 20892, USA.

ABSTRACT
Tooth and bone specimen require extensive demineralization for careful analysis of cell morphology, as well as gene and protein expression levels. The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate. This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest. To analyze LacZ activity, and the expression of other genes and their protein products in teeth and bones, it is necessary to carry out a complete demineralization of the specimen before cutting sections. However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase. Therefore, most protocols currently use mild acids such as 0.1 M ethylene diamine tetra-acetic acid (EDTA) for demineralization of tooth and bone specimen, which require a longer period of treatment for complete demineralization. A method by which hard tissue specimens such as teeth and bones can be rapidly, but gently, decalcified is necessary to save time and effort. Here, we report a suitable method for rapid demineralization of mouse teeth in 0.1M EDTA at 42˚C without any loss of ß-galactosidase activity.

No MeSH data available.


Related in: MedlinePlus