DOI 10.1007/s00418-012-0936-1SHORT COMMUNICATIONS Improved methods for detection of -galactosidase (lacZ) activity in hard tissue

coli is widely used as a reporter gene. The expression of lacZ can be detected by enzyme-based histochemical stain-ing using chromogenic substrates such as 5-bromo-4-chloro-3-indolyl--D-galactoside (X-gal). Because the enzymatic activity of lacZ is vulnerable to high tempera-tures and acid treatment for demineralization, detection of lacZ on paraYnized sections is diYcult, especially for hard tissues, which require demineralization before sectioning in paraYn. To circumvent this problem, whole-mount X-gal staining before sectioning is performed. However, detec-tion of lacZ activity in the center of larger portions of hard whole adult tissues is challenging. In this study, focusing on Wxation procedures, we determined the conditions con-ducive to improved detection of lacZ activity in deeper areas of whole tissues. We used an annexin a5 (Anxa5)-lacZ reporter mouse model in which the Anxa5 expression in hard tissue is indicated by lacZ activity. We found that lacZ activity could be detected throughout the periodontal liga-ment of adult mice when Wxed in 100 % acetone, whereas it was not detected in the periodontal ligament around the root apex Wxed in glutaraldehyde and paraformaldehyde. This staining could not be detected in wild-type mice. Ace-tone maintains the lacZ activity within 48 h of Wxation at both 4°C and at room temperature. In conclusion, acetone is the optimal Wxative to improve permeability for staining of lacZ activity in large volumes of adult hard tissues