IN-SITU HYBRIDIZATION IN SKELETAL TISSUES UTILIZING NONRADIOACTIVE PROBES

In situ hybridization (ISH) is a powerful technique that can be used t o localize specific DNA or RNA sequences to individual cells within a complex tissue. This combination of traditional morphology with molecu lar technology gives ISH an advantage over other types of molecular te chniques. Clinical and research applications of ISH have increased dra matically with the development of nonradioactive detection systems and chemical synthesis of oligonucleotide probes. Although ISH has the po tential to be a valuable tool for the study of bone diseases and norma l skeletal development, the mineralization of these tissues and the co nsequent need for decalcification prior to processing presents serious technical problems, In this paper we provide a detailed methodology f or performing ISH on skeletal tissues using synthetic oligonucleotide probes end-labeled with 11-dUTP-digoxigenin. Avian epiphyseal growth p lates that differentially express a variety of matrix proteins during maturation were examined. Data are presented on how our laboratory has optimized both the decalcification process and enzymatic treatment of this tissue to facilitate preservation of and access to nucleic acids . This method of ISH is both highly reproducible and technically feasi ble in pathology and orthopedics laboratories studying decalcified ske letal tissues.