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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 rapid demineralization at higher temperature.Soft x-ray images of the mouse skulls fixed with 4% PFA for 1 hour during the demineralization process at different temperatures; RT, 37°C, 42°C, and 50°C showing various degrees of tissue being decalcified at different time points, as evidenced by the steady decrease in radioopacity area. The far right panel in each row shows the incisor sections made from completely decalcified skull and stained with von Kossa to confirm complete demineralization, as indicated by the light brown precipitate. 0.1M EDTA is used as a demineralizeing agent for all of the skulls at indicated temperatures.
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Figure 4: The rapid demineralization at higher temperature.Soft x-ray images of the mouse skulls fixed with 4% PFA for 1 hour during the demineralization process at different temperatures; RT, 37°C, 42°C, and 50°C showing various degrees of tissue being decalcified at different time points, as evidenced by the steady decrease in radioopacity area. The far right panel in each row shows the incisor sections made from completely decalcified skull and stained with von Kossa to confirm complete demineralization, as indicated by the light brown precipitate. 0.1M EDTA is used as a demineralizeing agent for all of the skulls at indicated temperatures.

Mentions: Since moderate increase in incubation temperature could shorten the demineralization process time period for the analysis of β-galactosidase activity, we tested the effects of elevated temperatures during the demineralization process using 0.1M EDTA as a decalcifier. The degree of opacity was well-correlated with the degree of skull demineralization. The undecalcified areas of the skulls appeared as radio-opacity in the x-ray image. At RT, 10 days of incubation were required for complete demineralization, however just 6, 5, and 3 days were required at 37°C, 42°C, and 50°C, respectively (Fig. 4). Next, in order to test the effects of each temperature on the stability of β-galactosidase activity at the time of complete demineralization, we decalcified the skulls with 0.1M EDTA for a sufficient period of time, which was 10 days at RT, 6 days at 37°C, 5 days at 42°C, and 3 days at 50°C, and then we analyzed the β-galactosidase enzyme activity in the sections. We found that the skulls decalcified at 37°C and 42°C, but not at 50°C, showed equivalent β-galactosidase enzyme activity, although at 50°C the incubation time was the shortest (Fig. 5). The successful completion of the demineralization process was indicated by the light brown precipitate on tooth sections in Fig. (5b, d, fandh), as opposed to the dark brown calcium deposit shown in the undecalcified control tooth section (Fig. 5i).


A method for rapid demineralization of teeth and bones.

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

The rapid demineralization at higher temperature.Soft x-ray images of the mouse skulls fixed with 4% PFA for 1 hour during the demineralization process at different temperatures; RT, 37°C, 42°C, and 50°C showing various degrees of tissue being decalcified at different time points, as evidenced by the steady decrease in radioopacity area. The far right panel in each row shows the incisor sections made from completely decalcified skull and stained with von Kossa to confirm complete demineralization, as indicated by the light brown precipitate. 0.1M EDTA is used as a demineralizeing agent for all of the skulls at indicated temperatures.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: The rapid demineralization at higher temperature.Soft x-ray images of the mouse skulls fixed with 4% PFA for 1 hour during the demineralization process at different temperatures; RT, 37°C, 42°C, and 50°C showing various degrees of tissue being decalcified at different time points, as evidenced by the steady decrease in radioopacity area. The far right panel in each row shows the incisor sections made from completely decalcified skull and stained with von Kossa to confirm complete demineralization, as indicated by the light brown precipitate. 0.1M EDTA is used as a demineralizeing agent for all of the skulls at indicated temperatures.
Mentions: Since moderate increase in incubation temperature could shorten the demineralization process time period for the analysis of β-galactosidase activity, we tested the effects of elevated temperatures during the demineralization process using 0.1M EDTA as a decalcifier. The degree of opacity was well-correlated with the degree of skull demineralization. The undecalcified areas of the skulls appeared as radio-opacity in the x-ray image. At RT, 10 days of incubation were required for complete demineralization, however just 6, 5, and 3 days were required at 37°C, 42°C, and 50°C, respectively (Fig. 4). Next, in order to test the effects of each temperature on the stability of β-galactosidase activity at the time of complete demineralization, we decalcified the skulls with 0.1M EDTA for a sufficient period of time, which was 10 days at RT, 6 days at 37°C, 5 days at 42°C, and 3 days at 50°C, and then we analyzed the β-galactosidase enzyme activity in the sections. We found that the skulls decalcified at 37°C and 42°C, but not at 50°C, showed equivalent β-galactosidase enzyme activity, although at 50°C the incubation time was the shortest (Fig. 5). The successful completion of the demineralization process was indicated by the light brown precipitate on tooth sections in Fig. (5b, d, fandh), as opposed to the dark brown calcium deposit shown in the undecalcified control tooth section (Fig. 5i).

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