Method for detecting the expression of bone matrix protein by in situ hybridization using decalcified mineralized tissue

The sensitive and high resolution method of in situ hybridization technique has been developed using digoxigenin-11 UTP labeled cRNA as a probe. The system was applied to the decalcified mineralized tissues such as bone and dentin. Using the system, the localization of the mRNA of bone extracellular matrix proteins, osteopontin (Osp), osteonectin (Osn), Osteocalcin (Osc) and matrix Gla protein (MGP) was examined in decalcified bone. Decalcified femurs and mandibulae of embryo, neonatal, 2 to 40-week old mice were used for examination. Osn and Osc mRNAs were localized at osteoblast in bone and at odontoblast in dentin. Although the distribution pattern of Osn positive cells and Osc positive cells was partially overlapped, Osc mRNA was detected at matured osteoblast and odontoblast but Osn mRNA was detected not only at matured but differentiating osteoblast such as flat osteoblast in the periosteal layer. Osp mRNA was detected at osteoblast in bone, but no apparent expression of Osp mRNA was found at odontoblast in dentin. MGP mRNA was detected in hypertrophic chondrocytes. These results indicated the usefulness of this system for identifying the cell types in bone and dentin.