In situ hybridization for light and electron microscopy: a comparison of methods for the localization of viral RNA using biotinylated DNA and RNA probes.
We report procedures for in situ hybridization at the light and electron microscopic level for localization of viral RNA in poliovirus-infected, Lowicryl-embedded cells. We compare specificity and signal intensity of biotinylated, double-stranded DNA and single-stranded, strand-specific RNA probes, both corresponding to the same region of the poliovirus genome. The hybrids were detected with antibiotin antibodies or streptavidin with colloidal gold as a marker. Hybridization with the RNA probe was more sensitive and gave lower background than with DNA. Detection with immunogold proved to be by far more sensitive than with streptavidin. The hybridization and detection protocols for the DNA and the RNA probes could be applied without modification to light microscopic semi-thick sections as well as to electron microscopic ultrathin sections.
Troxler
M
M
Institute for Medical Microbiology, University of Basel, Switzerland.
Pasamontes
L
L
Egger
D
D
Bienz
K
K
D001710:Biotin; D002460:Cell Line; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis*; D006801:Humans; D008854:Microscopy, Electron / Q000379:methods; D009693:Nucleic Acid Hybridization; D017955:Poliovirus / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D015347:RNA Probes; D012367:RNA, Viral / Q000235:genetics; D014158:Transcription, Genetic
D015342:DNA Probes; D004279:DNA, Viral; D015347:RNA Probes; D012367:RNA, Viral; D001710:Biotin