Postembedding Ultrastructural in Situ Hybridization on Ultrathin Cryosections and LR White Resin Sections

Abstract

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.

Biotin and Digoxigenin as Labels for Light and Electron Microscopy in Situ Hybridization Probes: Where Do We Stand?

“…Bendayan et al (1984) have demonstrated elegantly that immunogold conjugates never enter the depth of a section but rather remain stuck at its surface, even when hydrophilic resins are used. Pretreatment of sections with etching reagents and digesting enzymes have been reported to improve hybridization efficiency in postembedding ISH (Morey 1995;Lin et al 1993;Mandry et al 1993;Puvion-Dutilleul and Pichard 1992;Wolber et al 1988). However, cell structures were damaged and nonspecific binding of gold particles increased, probably owing to surface roughness of sections engendered by etching (Yi et al 1995).…”