IN-SITU HYBRIDIZATION OF PARATHYROID HORMONE-RELATED PROTEIN IN NORMAL SKIN, SKIN TUMORS, AND GYNECOLOGICAL CANCERS USING DIGOXIGENIN-LABELED PROBES AND ANTIBODY ENHANCEMENT

We describe a novel procedure for in situ hybridization that combines the use of digoxigenin-labeled oligonucleotide probes with an antibody enhancement step that can be performed on formalin-fixed, paraffin-em bedded tissues. Addition of a second antibody enhances the visibility of parathyroid hormone-related protein (PTHrP) mRNA expression from ba rely to highly discernible and interpretable, with virtually no nonspe cific background expression. This technique has allowed visualization of PTHrP mRNA in normal human skin and epithelium-derived tumors. PTHr P mRNA expression was confined to the basal and spinous keratinocyte l ayers of skin. There was strong hybridization in the spinous keratinoc yte layer and a low level of hybridization in the basal layer. An exte nsive panel of positive and negative controls included poly d(T) probe to indicate total mRNA present in the sections. Squamous cell carcino mas and basal cell carcinomas of the skin, from pathology archives, we re examined for the presence of PTHrP mRNA. The results reflected prev ious immunohistochemical studies, with every squamous cell carcinoma h ybridizing strongly with the PTHrP probes. The basal cell carcinomas s howed no expression of PTHrP mRNA, although the total mRNA signal was very strong. The localization of PTHrP mRNA in the tumors of the gynec ological tract also reflected the immunohistochemical findings, with e xpression found in the squamous cell carcinomas but not in the adenoca rcinomas. In situ hybridization with digoxigenin-labeled oligonucleoti de probes and antibody enhancement has provided a sensitive, highly sp ecific procedure for detection of PTHrP mRNA in tumors and normal tiss ue.