In situ hybridization at light and electron microscopic levels: identification of human papillomavirus nucleic acids.

A comparison of the sensitivities of biotinylated and 32P-labelled human papillomavirus type 6b DNA probes was made. Slot blot hybridization results showed that the sensitivity of biotinylated probes was consistent with that of 32P-labelled, that is, 0.1 pg of pBR 322 plasmid containing 8 kbp HPV cDNA. In situ hybridization using 35S-labelled probes was applied to tissue from condylomata acuminata. After autoradiography, many silver grains were seen concentrated over the superficial koilocytic nuclei with some grains present in the cytoplasm. Biotinylated probes were visualized by 4 different means, i.e., streptavidin alkaline phosphatase, streptavidin biotinylated horseradish peroxidase, monoclonal anti-biotin antibody with 15 nm colloidal gold and streptavidin 5 nm colloidal gold. Strong reaction products were localized in the superficial nuclei while the cytoplasm of koilocytes showed weak hybridization signal. Pre-embedding methods were carried out for electron microscopic studies in which numerous granular diaminobenzidine (DAB) products were present in the nuclear chromatin while viral particles themselves had much fewer DAB products. This suggested that hybridization occurred more efficiently to yet unassembled viral genomes than to matured virions. Post-embedding methods using 15 nm colloidal gold were performed and showed singly scattered or clustered gold particles in superficial koilocytic nuclei.