An improved procedure of electron microscopic in situ hybridization for detecting adenovirus DNA.
Electron microscopic in situ hybridization (EM-ISH) is a useful method in determining the localization of a specific nucleic acid at the ultrastructural level. Since the EM-ISH protocol includes many steps, no standard protocol for EM-ISH is available yet. In this study, we optimized quantitatively the critical conditions with respect to embedding resin, nucleic acid labeling and hybridization reaction time, by using adenovirus-infected cells as the indicator cells. The optimal detection of an adenovirus-specific nucleic acid was obtained by overnight hybridization reaction on sections embedded in Lowicryl K4M resin. Random-primed-labeled probes improved the reactivity. At least 60% of virus particles in paracrystalline arrays was found to contain viral DNA. These arrays in adenovirus-infected cells are useful in evaluating quantitatively the efficiency of protocols of EM-ISH.
Goto
T
T
Kohno
T
T
Nakano
T
T
Fujioka
Y
Y
Morita
C
C
Sano
K
K
D000256:Adenoviridae / Q000648:ultrastructure*; D000818:Animals; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000648:ultrastructure*; D017403:In Situ Hybridization / Q000379:methods*; D008854:Microscopy, Electron / Q000379:methods*; D014709:Vero Cells
D004279:DNA, Viral