Modified protocol for accelerated decalcification of mouse teeth

Objectives: Detection of gene expression in hard tissues requires an extensive decalcification. It is critical to preserve the mRNA and in the case of transgenic animals that carry reporter genes such as E. coli beta-galactosidase (lacZ), the enzyme activity for precise localization. The current decalcification procedure requires an incubation period of 3-4 weeks at room temperature. In order to detect the gene expression profiles in a relatively short time, we have modified and developed an efficient method to accelerate the decalcification of skulls from mice of various ages. Methods: We have used the dspp-lacZ transgenic mouse for these studies. The skulls from these mice of different ages were cut in half, fixed in either buffered formalin, 4% paraformaldehyde, or 0.25% glutaraldehyde, decalcified using either acid-based decalcification agents (formical and immunocal) or an EDTA-based agent over a linear period of time at different temperatures. Decalcification was verified by X-ray analysis and with von Kossa's staining. The effect of decalcification on tooth proteins was analyzed by lacZ activity on frozen sections. Results: Paraformaldehyde and glutaraldehyde were found to be the most efficient fixative agents. Decalcification was faster with the EDTA-based agent at 42^oC without compromising the lacZ staining in the tooth from skulls fixed with either PFA or glutaraldehyde. Most importantly, decalcification carried out at 42^oC significantly shortened incubation time. Conclusions: Our modified protocol can effectively decalcify mouse skulls in a shorter period of time without compromising stability and activity of the reporter enzyme.