In situ hybridisation in formalin fixed, paraffin wax embedded liver specimens: method for detecting human and viral DNA using biotinylated probes

Abstract

A rapid and reproducible technique for in situ hybridisation, using biotin labelled probes for the Y chromosome, human DNA, hepatitis B virus DNA and cytomegalovirus DNA on formalin fixed, paraffin embedded liver tissue, was developed. The degree of proteolytic digestion of tissue specimens is critical to ensure adequate unmasking of DNA and to avoid non-specific staining, a consequence of endogenous biotin in liver. Specific in situ hybridisation was achieved after digestion with pepsin, proteinase K, or protease, which gave optimal results. Both hepatitis B virus DNA and cytomegalovirus DNA were visualised in tissue from patients with chronic hepatitis B virus infection or in liver transplant recipients, respectively; the distribution of viral DNA was shown to be quite distinct between the two groups of patients.