Ultrastructural localization of defined sequences of viral RNA and DNA by in situ hybridization of biotinylated DNA probes on sections of herpes simplex virus type 1 infected cells

Abstract

The influence of fixation and enzymatic digestions on the ability of a denatured double-stranded DNA probe to bind specifically to related sequences of RNA and DNA in sections of Lowicryl embedded cells was investigated. Specificity of the hybridization was assessed using a biotinylated cloned subgenomic herpes simplex virus type 1 DNA fragment to localize viral nucleic acids in sections of infected cells. The probe was detected by anti-biotin antibodies and indirect immunogold labeling. Controls indicated that protease digestion of proteins from the section eliminated non-specific binding of the probe and labeling of endogenous biotin. Both formaldehyde and glutaraldehyde fixation retained viral RNA in protease digested sections. Its labeling was randomly and sparsely distributed over the fibrillo-granular network of the infected nucleus and over the ribosome-rich regions of cytoplasm. Labeling of single-stranded portions of viral DNA in protease-RNase digested sections was infrequent. It was located precisely over nucleoids of a few viral nucleocapsids whatever their location in the cell and their stage of maturation. Labeling of double-stranded viral DNA by denaturation of the DNA in the sections of Lowicryl embedded cells was possible after fixation with formaldehyde but not glutaraldehyde. Among several denaturation protocols, 0.5 N NaOH treatment was best for hybridization of both non-encapsidated and encapsidated viral DNA in protease-RNase digested sections. Free viral genomes were detected exclusively within the virus-replicating region of infected nuclei. Labeling of viral nucleoids was independent of their location in the cell. The high percentage of labeled viral nucleoids suggests that the related viral DNA sequence is not aggregated in the nucleoid but is extended and therefore numerous portions of this defined DNA sequence are accessible at the surface of the section for the binding of the probe.

Highly Ordered Spatial Organization of the Structural Long Noncoding NEAT1 RNAs within Paraspeckle Nuclear Bodies

“…Hybrids were detected with a goat anti-biotin antibody conjugated to 10-nm gold particles (BBInternational). Detection of DNA requires NaOH treatment of the thin sections before hybridization (Puvion-Dutilleul and Puvion, 1991; Puvion-Dutilleul and Pierron, 1992). This treatment was omitted in all our experiments.…”

Detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp by in situ hybridization at the electron microscope level

“…Fixation with glutaraldehyde concentrations higher than 1% has been counter-indicated for specimens subjected to in situ hybridization for electron microscopy (Le Guellec 1998) because of interference with the localization of double-stranded DNA. However, hybridization with single-stranded DNA is possible, especially after proteolytic (enzymatic) digestion (Puvion & Puvion 1991). Indeed, the Proteinase K step increased intensity of the in situ hybridization reaction, suggesting that excessive DNA to protein cross-links had been removed.…”

Intracellular localization of parvovirus B19 nucleic acid at the ultrastructural level by in situ hybridization with digoxigenin-labelled probes

“…A recurring problem with the use of biotinylated probes for in situ hybridization at the light-microscope HIST 25/(~-B level is the presence of endogenous biotin leading to false-positive signal. One report has indicated that this can also be a problem at the ultrastructural level unless proteolytic digestion is performed (Puvion-Dutilleul & Puvion, 1991). Digoxigenin is gaining in popularity as an alternative non-isotopic probe label at the light microscope level, being capable of providing high resolution localization of target sequences with minimal background signal .…”