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. 1992 Dec;45(12):1099–1104. doi: 10.1136/jcp.45.12.1099

In situ hybridisation of EBV DNA-DNA hybrids using wet heat in polypropylene containers.

L G Labrecque 1
PMCID: PMC495005  PMID: 1336018

Abstract

AIMS: To explore procedures designed to optimise DNA-DNA in situ hybridisation, using cells infected with Epstein-Barr virus (EBV) and tissues and subfragments of the EBV DNA as probes. METHODS: The denaturation step occurred in a polypropylene container, using wet heat generated by a hot water container, the pressure cooker, or the microwave oven, without coverslips, reaching a temperature of 121 degrees C or more in these two last systems. Two different visualisation systems were used. RESULTS: Fixed cells and tumours harbouring a high and medium to low copy number (a few hundreds to 33 copies per cell), were clearly labelled, using a simple reiterated subfragment (BamW) of the EBV DNA, and fresh frozen cells, harbouring a very low copy number (one to two on average) labelled using BamW as well as BamH (single non-reiterated 6 kilobase subfragment). CONCLUSION: This is a valuable alternative technique for DNA-DNA ISH that can be used in fresh frozen samples as well as fixed samples.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brigati D. J., Myerson D., Leary J. J., Spalholz B., Travis S. Z., Fong C. K., Hsiung G. D., Ward D. C. Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes. Virology. 1983 Apr 15;126(1):32–50. doi: 10.1016/0042-6822(83)90460-9. [DOI] [PubMed] [Google Scholar]
  2. Buongiorno-Nardelli M., Amaldi F. Autoradiographic detection of molecular hybrids between RNA and DNA in tissue sections. Nature. 1970 Mar 7;225(5236):946–948. doi: 10.1038/225946a0. [DOI] [PubMed] [Google Scholar]
  3. Burns J., Chan V. T., Jonasson J. A., Fleming K. A., Taylor S., McGee J. O. Sensitive system for visualising biotinylated DNA probes hybridised in situ: rapid sex determination of intact cells. J Clin Pathol. 1985 Oct;38(10):1085–1092. doi: 10.1136/jcp.38.10.1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Coates P. J., Hall P. A., Butler M. G., D'Ardenne A. J. Rapid technique of DNA-DNA in situ hybridisation on formalin fixed tissue sections using microwave irradiation. J Clin Pathol. 1987 Aug;40(8):865–869. doi: 10.1136/jcp.40.8.865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cremers A. F., Jansen in de Wal N., Wiegant J., Dirks R. W., Weisbeek P., van der Ploeg M., Landegent J. E. Non-radioactive in situ hybridization. A comparison of several immunocytochemical detection systems using reflection-contrast and electron microscopy. Histochemistry. 1987;86(6):609–615. doi: 10.1007/BF00489555. [DOI] [PubMed] [Google Scholar]
  6. Gall J. G., Pardue M. L. Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci U S A. 1969 Jun;63(2):378–383. doi: 10.1073/pnas.63.2.378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hitt M. M., Allday M. J., Hara T., Karran L., Jones M. D., Busson P., Tursz T., Ernberg I., Griffin B. E. EBV gene expression in an NPC-related tumour. EMBO J. 1989 Sep;8(9):2639–2651. doi: 10.1002/j.1460-2075.1989.tb08404.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Huang W. M., Gibson S. J., Facer P., Gu J., Polak J. M. Improved section adhesion for immunocytochemistry using high molecular weight polymers of L-lysine as a slide coating. Histochemistry. 1983;77(2):275–279. doi: 10.1007/BF00506570. [DOI] [PubMed] [Google Scholar]
  9. John H. A., Birnstiel M. L., Jones K. W. RNA-DNA hybrids at the cytological level. Nature. 1969 Aug 9;223(5206):582–587. doi: 10.1038/223582a0. [DOI] [PubMed] [Google Scholar]
  10. MaWhinney W. H., Warford A., Rae M. J., Lauder I. Automated immunochemistry. J Clin Pathol. 1990 Jul;43(7):591–596. doi: 10.1136/jcp.43.7.591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Manuelidis L., Langer-Safer P. R., Ward D. C. High-resolution mapping of satellite DNA using biotin-labeled DNA probes. J Cell Biol. 1982 Nov;95(2 Pt 1):619–625. doi: 10.1083/jcb.95.2.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Morris R. G., Arends M. J., Bishop P. E., Sizer K., Duvall E., Bird C. C. Sensitivity of digoxigenin and biotin labelled probes for detection of human papillomavirus by in situ hybridisation. J Clin Pathol. 1990 Oct;43(10):800–805. doi: 10.1136/jcp.43.10.800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Stark E., Faltinat D., Von der Fecht R. An automated device for immunocytochemistry. J Immunol Methods. 1988 Feb 24;107(1):89–92. doi: 10.1016/0022-1759(88)90013-0. [DOI] [PubMed] [Google Scholar]
  14. Stross W. P., Jones M., Mason D. Y. Automation of APAAP immunocytochemical technique. J Clin Pathol. 1989 Jan;42(1):106–112. doi: 10.1136/jcp.42.1.106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Takahashi T., Ishiguro K. Development of an automatic machine for in situ hybridization and immunohistochemistry. Anal Biochem. 1991 Aug 1;196(2):390–402. doi: 10.1016/0003-2697(91)90484-b. [DOI] [PubMed] [Google Scholar]
  16. Teo C. G., Griffin B. E. Visualization of single copies of the Epstein-Barr virus genome by in situ hybridization. Anal Biochem. 1990 Apr;186(1):78–85. doi: 10.1016/0003-2697(90)90576-u. [DOI] [PubMed] [Google Scholar]
  17. Unger E. R., Budgeon L. R., Myerson D., Brigati D. J. Viral diagnosis by in situ hybridization. Description of a rapid simplified colorimetric method. Am J Surg Pathol. 1986 Jan;10(1):1–8. doi: 10.1097/00000478-198601000-00001. [DOI] [PubMed] [Google Scholar]

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