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

Effects of fixative solutions on β-galactosidase activity.The cross sections of the mandibular incisors of the 3-month-old Dspp-LacZ mouse skulls fixed with either 0.25% Glutaraldehyde (a), 4% paraformaldehyde (b), Zinc Formalin (c), or 10% Formalin (d), for 1 h at RT, decalcified in 0.1% EDTA for 3 weeks and then stained for β-galactosidase activity. In all the sections, clear staining was observed in the odontoblast layer and some positive spots were detected in the tooth pulp. d, dentin; p, pulp.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Effects of fixative solutions on β-galactosidase activity.The cross sections of the mandibular incisors of the 3-month-old Dspp-LacZ mouse skulls fixed with either 0.25% Glutaraldehyde (a), 4% paraformaldehyde (b), Zinc Formalin (c), or 10% Formalin (d), for 1 h at RT, decalcified in 0.1% EDTA for 3 weeks and then stained for β-galactosidase activity. In all the sections, clear staining was observed in the odontoblast layer and some positive spots were detected in the tooth pulp. d, dentin; p, pulp.

Mentions: Evaluation of fixatives and fixation time on β-galactosidase activity: The effects of different fixatives on β-galactosidase activity in soft tissues were reported earlier [17], however there are no such reports about the effects of different fixatives on hard tissues such as teeth. To examine which fixative agent is suitable to analyze β-galactosidase activity in tooth sections, we first compared the effects of 1 hour fixation by 0.25% glutaraldehyde, 4% PFA, zinc formalin, and formalin. As shown in Fig. (1), we could detect similar levels of β-galactosidase activity in the odontoblasts of the incisors from the skulls fixed with different fixatives, indicating that treatment with these agents retained the enzyme activity at a similar level. To further investigate the optimum fixation time for analyzing β-galactosidase activity in teeth, we post-fixed the tooth section with 4% PFA for different periods of times. All the time periods tested, except for the 2 hour time period, yielded a strong signal for β-galactosidase activity (Fig. 2).


A method for rapid demineralization of teeth and bones.

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

Effects of fixative solutions on β-galactosidase activity.The cross sections of the mandibular incisors of the 3-month-old Dspp-LacZ mouse skulls fixed with either 0.25% Glutaraldehyde (a), 4% paraformaldehyde (b), Zinc Formalin (c), or 10% Formalin (d), for 1 h at RT, decalcified in 0.1% EDTA for 3 weeks and then stained for β-galactosidase activity. In all the sections, clear staining was observed in the odontoblast layer and some positive spots were detected in the tooth pulp. d, dentin; p, pulp.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Effects of fixative solutions on β-galactosidase activity.The cross sections of the mandibular incisors of the 3-month-old Dspp-LacZ mouse skulls fixed with either 0.25% Glutaraldehyde (a), 4% paraformaldehyde (b), Zinc Formalin (c), or 10% Formalin (d), for 1 h at RT, decalcified in 0.1% EDTA for 3 weeks and then stained for β-galactosidase activity. In all the sections, clear staining was observed in the odontoblast layer and some positive spots were detected in the tooth pulp. d, dentin; p, pulp.
Mentions: Evaluation of fixatives and fixation time on β-galactosidase activity: The effects of different fixatives on β-galactosidase activity in soft tissues were reported earlier [17], however there are no such reports about the effects of different fixatives on hard tissues such as teeth. To examine which fixative agent is suitable to analyze β-galactosidase activity in tooth sections, we first compared the effects of 1 hour fixation by 0.25% glutaraldehyde, 4% PFA, zinc formalin, and formalin. As shown in Fig. (1), we could detect similar levels of β-galactosidase activity in the odontoblasts of the incisors from the skulls fixed with different fixatives, indicating that treatment with these agents retained the enzyme activity at a similar level. To further investigate the optimum fixation time for analyzing β-galactosidase activity in teeth, we post-fixed the tooth section with 4% PFA for different periods of times. All the time periods tested, except for the 2 hour time period, yielded a strong signal for β-galactosidase activity (Fig. 2).

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