In Situ Hybridization for Detection of Viral Nucleic Acid in Cell Cultures and Tissues

1986 ◽  
pp. 203-223 ◽  
Author(s):  
Howard E. Gendelman ◽  
Scott Koenig ◽  
Allen Aksamit ◽  
Sundarajan Venkatesan ◽  
Wallace W. Tourtellotte ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Cell Cultures ◽  
Viral Nucleic Acid

scholarly journals Distribution of Bluetongue Virus in Tissues of Experimentally Infected Pregnant Dogs as Determined by In Situ Hybridization

1996 ◽  
Vol 33 (3) ◽  
pp. 337-340 ◽  
Author(s):  
C. C. Brown ◽  
J. C. Rhyan ◽  
M. J. Grubman ◽  
L. A. Wilbur
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Bluetongue Virus ◽  
Mononuclear Cells ◽  
Nonstructural Protein ◽  
The Other ◽  
Post Inoculation ◽  
Viral Nucleic Acid ◽  
Nonstructural Protein 1

Six female dogs (four pregnant and two nonpregnant) were inoculated with bluetongue virus (BTV), serotype 11. Pregnant animals and one nonpregnant dog received 5.5-6.3 log10 of cell culture-adapted virus. The other nonpregnant dog received a modified live vaccine contaminated with bluetongue virus. The nonpregnant animals never became clinically ill and were euthanatized 35 days post-inoculation. Three of the four pregnant dogs aborted, and all four died or were euthanatized 5-10 days post-inoculation. The predominant pathologic feature in the adults was severe pulmonary edema. Various tissues from the bitches and fetuses were examined by in situ hybridization using a digoxigenin-labeled probe corresponding to the nonstructural protein-1 gene of BTV-17. By this technique, viral nucleic acid was detected predominantly in endothelial cells of lung of all four dogs, with lesser amounts in capillaries of uterus, spleen, and kidney in some of the dogs. In two adult dogs, bluetongue viral nucleic acid was detected in mononuclear cells of the periarteriolar lymphoid sheaths of spleen. There was minimal staining of capillaries in placentae in three of the five fetuses examined. There was no viral nucleic acid detected in any of the other fetal tissues.

scholarly journals Comparative usefulness of tissue fixatives for in situ viral nucleic acid hybridization.

1985 ◽  
Vol 33 (10) ◽  
pp. 1026-1032 ◽  
Author(s):  
H A McAllister ◽  
D L Rock
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Pseudorabies Virus ◽  
Nucleic Acid Hybridization ◽  
Viral Nucleic Acid ◽  
Tissue Sections ◽  
Laboratory Rodents ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded

Traditionally tissues for in situ hybridization of viral nucleic acid have been small pieces obtained from laboratory rodents, and fixatives that are designed for electron microscopy, such as periodate-lysine-paraformaldehyde (PLP) can handle them adequately. However, these fixatives have limited penetrating ability and may produce no appreciable hardening, so alternative fixation methods were evaluated. The intention was to determine whether fixatives adequate for bulky tissues such as whole or halved pig and cow brains would also be compatible with in situ hybridization. Various fixatives were evaluated using a system of intracranial inoculation of BALB/c mice with pseudorabies virus (PRV) followed by in situ hybridization of brain tissue sections with a 35S-labeled PRV DNA probe. Loss of tissue sections was a major problem, particularly with PLP and formalin, but positive results were obtained with five fixatives tested. Cellular morphology was especially good with PLP and with a modification of Carnoy's fluid, MOCA fixative. An incidental but important observation was that formalin is compatible with in situ hybridization. Retroactive studies of viral diseases using routinely processed blocks of tissue (formalin-fixed, paraffin-embedded) are therefore conceivable.

scholarly journals The natural history of encephalomyocarditis virus-induced myositis and myocarditis in mice. Viral persistence demonstrated by in situ hybridization.

1988 ◽  
Vol 168 (5) ◽  
pp. 1639-1648 ◽  
Author(s):  
M E Cronin ◽  
L A Love ◽  
F W Miller ◽  
P R McClintock ◽  
P H Plotz
Keyword(s):  
Skeletal Muscle ◽  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Single Cells ◽  
Viral Persistence ◽  
Encephalomyocarditis Virus ◽  
High Yield ◽  
Viral Nucleic Acid ◽  
History Of

Picornaviruses can initiate chronic inflammation that persists after the virus can no longer be cultured from inflamed tissues. In an attempt to understand this transition we have sought evidence for viral persistence by methods that detect viral genome independent of whether or not whole competent virus is present. In mice infected with a myotropic variant of encephalomyocarditis virus, EMC-221A, virus can be cultured in high yield at 1 wk and in low yield at 2 wk from skeletal muscle, heart, and brain; a small number of plaque-forming units could be cultured from brain at 4 wk. By contrast, in situ hybridization detected viral nucleic acid at least a week or two thereafter, often in single cells. In the skeletal muscle, inflammation disappeared by 3 wk, but in heart it remained for the full 12 wk of observation. In the brain, microglial nodules, sometimes with associated viral nucleic acid, were present for a long period. Application of this technique allows a more accurate assessment of the role of viral persistence in the pathogenesis of virus-initiated but apparently autoimmune inflammation.

scholarly journals Locked Nucleic Acid In situ Hybridization Analysis of miR-21 Expression during Colorectal Cancer Development

2009 ◽  
Vol 15 (12) ◽  
pp. 4009-4016 ◽  
Author(s):  
Nobutake Yamamichi ◽  
Ryoichi Shimomura ◽  
Ken-ichi Inada ◽  
Kouhei Sakurai ◽  
Takeshi Haraguchi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Hybridization Analysis ◽  
Locked Nucleic Acid ◽  
Cancer Development

scholarly journals Improved In Situ Hybridization Efficiency with Locked-Nucleic-Acid-Incorporated DNA Probes

2006 ◽  
Vol 72 (8) ◽  
pp. 5311-5317 ◽  
Author(s):  
Kengo Kubota ◽  
Akiyoshi Ohashi ◽  
Hiroyuki Imachi ◽  
Hideki Harada
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Fluorescence Intensity ◽  
Dna Probes ◽  
Locked Nucleic Acid ◽  
Factors Affecting ◽  
Hybridization Efficiency ◽  
Probe Hybridization ◽  
Major Factors

ABSTRACT Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency.

scholarly journals Influence of prolonged formalin fixation of tissue samples on the sensitivity of chromogenic in situ hybridization

2011 ◽  
Vol 23 (6) ◽  
pp. 1212-1216 ◽  
Author(s):  
Meike M. Mostegl ◽  
Barbara Richter ◽  
Nora Dinhopl ◽  
Herbert Weissenböck
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Signal Intensity ◽  
Formalin Fixation ◽  
Proteinase K ◽  
Cross Linking ◽  
Fixation Time ◽  
Tissue Samples ◽  
Chromogenic In Situ Hybridization

Chromogenic in situ hybridization (ISH) is a commonly used tool in diagnostic pathology to detect pathogens in formalin-fixed, paraffin-embedded (FFPE) tissue sections. Prolonged formalin fixation time was identified to be a limiting factor for the successful detection of nucleic acid from different pathogens, most probably due to the cross-linking activity of formalin between RNA, DNA, and proteins. Therefore, in the current study, the influence of formalin fixation time on ISH signal intensity of 2 viral ( Porcine circovirus-2 [PCV-2] and Porcine respiratory and reproductive virus [PRRSV]) and 2 protozoal agents ( Cryptosporidium serpentis and Tritrichomonas sp.) was evaluated. Tissue samples were fixed in 7% neutral buffered formaldehyde solution, and at defined intervals, pieces were embedded in paraffin wax and subjected to pathogen-specific ISH. For all 4 pathogens, the signal intensity remained comparable with the starting ISH signal for different periods of fixation (PCV-2: 6 weeks, PRRSV: 23 weeks, C. serpentis: 55 weeks, Tritrichomonas sp.: 53 weeks). Thereafter, the signal started to decline until loss of nucleic acid detection. The influence of increased proteinase K concentrations for inverting the formalin-induced cross-linking activity was examined compared with the standard protocol. With all 4 infectious agents, a 4-fold proteinase K concentration restored the ISH signals to a level comparable with 1 day of fixation. In conclusion, the influence of prolonged formalin fixation on the intensity of detected ISH signal highly depends on the analyzed infectious agent and the pretreatment protocol.

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