Development and its application of simple methods for bone sections

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Dr.Yoshiki, a principal investigator of this research project, reported the simple method to identify osteoid tissues in decalcified bone sections. In this method, treatment of bone tissues with cyanuric chloride before decalcification is essential to identify osteoid tissues. In the present study, we conducted several experiments to develop new application of this technique for bone research, and obtained the following results.1) When tartrate acid resistant acid phosphatase (TRACP) activity was detected in various tissues prepared by conventional technique, its activity was found is not only osteoclasts but also mononuclear cells in spleen and lungs. However, its activity was detected in only osteoclasts when tissues were prepared after cyanuric chloride treatment. These results indicate that this technique is useful to identify osteoclast cell lineage in decalcified bone sections.2) We recently developed in situ hybridization technique for bone sections. We examined the effects of cyanuric chloride treatment on in situ hybridization. The expression of mRNAs for osteocalcin and type I collagen is detected in bone sections prepared after cyanuric chloride treatment as well as bone sections prepared by conventional technique. These results suggest that cyanuric chloride treatment dose not affect detection of mRNA by in situ hybridization. This also suggests the possibility of combined technique of histochemistry and in situ hybridization.3) We tried immunohistochemistry using bone sections treated with cyanuric chloride, but we obtained strong background staining. This technique is not useful for immunohistochemistry.