Postembedding ultrastructural in situ hybridization on ultrathin cryosections and LR white resin sections

A method was developed for nonisotopic postembedding in situ hybridization (ISH) on ultrathin sections of frozen and of LR White resin-embedded material at the electron microscopic level. The method was successfully applied to detect Epstein-Barr virus (EBV) DNA in the P3HR1 human Burkitt's lymphoma cell line. Each of the steps in the procedure had to be optimized for successful ISH on the frozen and LR White sections. The most important conditions are described. Predigestion with proteinase K was only necessary with the resin sections. Sections were treated with sodium hydroxide to denature target DNA and were hybridized with a biotinylated probe. The probe was best detected with a primary antibody to biotin followed by a gold-conjugated secondary antibody. EBV DNA was detected in the nucleus and/or cytoplasm in 10% to 20% of P3HR1 cells. A similar percentage of cells in thin L-sectioned material prepared by routine methods showed virus particles at different stages of maturation.