- Abstract To understand the pathogenesis of HBV infection at the single cell level, we have established a sensitive, specific and reproducible method for the simultaneous in situ detection of HBV-specific nucleotide sequences and antigens in paraffin-embedded liver tissue using nonradioactive hybridization and immunohistochemical techniques. Liver sections were stained for HBsAg or HBcAg by immunohistochemistry (IHC) using an enhanced biotin-streptavidin technique. Following immunohistochemical staining of the liver sections, in situ hybridization (ISH) was performed on the same section using a digoxigenin-labeled, HBV-specific probe. Specific hybridization was detected using an anti-digoxigenin, Fab fragment-alkaline phosphatase conjugate. This simultaneous assay permits the subcellular localization of HBV DNA and antigens with excellent preservation of tissue morphology and absence of background staining. In addition, the types and percentage of cells harboring HBV in the tissue can be determined. Although both reactions are detected by immunohistochemical methods, the application of a dual detection system using two different color reagents permits the identification of HBV antigens as a red signal and HBV DNA as a blue-purple signal at the single cell level. Both ISH and IHC can be performed in the same tissue section without significantly reducing the sensitivity of either assay. In addition, since this simultaneous detection assay can be completed within two days and eliminates the need for radioactive probes, it may be used effectively as a routine clinical procedure.