Post-embedding in Situ Hybridization for Localization of Viral Nucleic Acid in Ultra-thin Sections

We developed an in situ hybridization technique for localization of plant virus RNA in ultra-thin sections of virus-infected, Lowicryl HM20- or osmium-fixed, Araldite-embedded plant leaf tissues. A digoxigenin-labeled in vitro transcript corresponding to 173 nucleotides at the 3' end of bamboo mosaic virus (BaMV) RNA was used as a riboprobe and the hybrids were detected by incubation with sheep antidigoxigenin antibody followed by gold-labeled rabbit anti-sheep IgG. Pre-treatment of ultra-thin sections with an etching agent was the critical step in enhancing hybridization signals on Lowicryl-embedded thin sections. Etching combined with K-metaperiodate treatment increased the accessibility of nucleic acids to riboprobes in osmicated Araldite-embedded sections. BaMV RNA was specifically detected within chloroplasts, mitochondria, and nuclei of infected cells. BaMV virions and BaMV-specific electron-dense crystalline bodies were also labeled. The labeling intensity on Lowicryl-embedded samples, in general, was much higher than that on osmicated Araldite-embedded samples. However, our procedure offers the advantage that it permits labeling of viral nucleic acids in tissues already processed for routine EM and should be applicable for in situ labeling of any cellular nucleic acids.