Skip to main content
SpringerLink
Log in

The Technique of In Situ Hybridization

  • Protocol
Human Airway Inflammation

Part of the book series: Methods in Molecular Medicine ((MIMM,volume 56))

Abstract

In situ hybridization (ISH) (also called “hybridization histochemistry” or “hybridization cytology”) was first described in 1969 by Gall and Pardue who used the technique to localize ribosomal DNA in Xenopus oocytes (1). In contrast to other techniques of hybridization (i.e., Northern or Southern blotting), ISH allows detection of specific DNA or RNA molecules in single cells, histological sections, or chromosomes. During the past decades, ISH has become a powerful tool in molecular biology and pathology, and has been widely applied to the localization of viral DNA, detection of expression of messenger RNA, and analysis of genes in chromosomes. This chapter describes the technique of ISH in some detail and also explains the pitfalls associated with its use.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Gall, J. G. and Pardue, M. L. (1969) Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc. Natl. Acad. Sci. USA. 63, 378–381.

    Article  CAS  PubMed  Google Scholar 

  2. Feinberg, A. P. and Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13.

    Article  CAS  PubMed  Google Scholar 

  3. Rigby, P. W. J., Diekmann, M., Rhodes, C., and Berg, P. (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237–251.

    Article  CAS  PubMed  Google Scholar 

  4. Stahl, W. L., Eakin, T. J., and Baskin, D. G. (1993) Selection of oligonucleotide probe for detection of mRNA isoforms. J. Histochem. Cytochem. 41, 1735–1740.

    CAS  PubMed  Google Scholar 

  5. Lathe, R. (1985) Synthetic oligonucleotide probes deduced from amino acid sequences data: theoretical and practical considerations. J. Mol. Biol. 183, 1–12.

    Article  CAS  PubMed  Google Scholar 

  6. Gyllensten, U. B. and Erlin, H. A. (1988) Generation of single stranded DNA by the polymerase chain reaction and its application of direct sequencing of the HLA-DQA locus. Proc. Natl. Acad. Sci. USA. 83, 7652–7656.

    Article  Google Scholar 

  7. Cone, R. W. and Schlaepfer, E. (1998) Improved in situ hybridization to HIV with RNA probes derived from PCR products. J. Histochem. Cytochem. 45, 721–727.

    Google Scholar 

  8. Cox, K. H., DeLeon, D. V., Angerer, L. M., and Angerer, R. C. (1984) Detection of mRNAs in Sea Urchin embryos by in situ hybridization using asymmetric RNA probes. Develop Biol. 101, 485–502.

    Article  CAS  PubMed  Google Scholar 

  9. Melton, D. A., Krieg, P. A., and Rebagliati, M. R. (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acid Res. 12, 7035–7041.

    Article  CAS  PubMed  Google Scholar 

  10. Schenborn, E. T. and Mierendorf, R. C. Jr. (1985) A novel transcription property of SP6 and T7 RNA polymerase: dependence on template structure. Nucleic Acid Res. 13, 6226–6230.

    Article  Google Scholar 

  11. Young, I. D., Ailles, L., Deugan, K., and Kisilevsky, R. (1991) Transcription of cRNA for in situ hybridization from polymerase chain reaction-amplified DNA. Lab. Invest. 64, 709–712.

    CAS  PubMed  Google Scholar 

  12. Allen, J. M., Sasek, C. A., Martin, J. B., and Heinrich, G. (1987) Use of complementary 125I labeled RNA for single cell resolution by in situ hybridization. Biotechniques 5, 774–777.

    CAS  Google Scholar 

  13. Bakkenist, C. J. and McGee J. O’D. (1998) The preparation of non-radioisotopic hybridization probes, inIn Situ Hybridization (Polak, J. M. and McGee, J. O’D. eds) Oxford University Press, pp. 35–48.

    Google Scholar 

  14. Mcallister, H. A. and Rock, D. L. (1985) Comparative usefulness of tissue fixatives for in situ viral nucleic acid hybridization. J. Histochem. Cytochem. 33, 1026–1032.

    CAS  PubMed  Google Scholar 

  15. Bresser, J. and Evinger-Hodges, M. J. (1992) Comparison and optimization of in situ hybridization procedures yielding rapid sensitive mRNA detections. Gene. Anal. Tech. 4, 89–104.

    Article  Google Scholar 

  16. Ying, S., Robinson, D. S., Varney, V., Meng, Q., Tsicopoulos, A., Moqbel, R., et al. (1991) TNF-alpha mRNA expression in allergic Inflammation. Clin. Exp. Allergy. 21, 745–750.

    Article  CAS  PubMed  Google Scholar 

  17. Ying, S., Durham, S. D., Barkans, J., Masuyama, K., Jacobson, M., Rak, S., Lowhagen, O., et al. (1993) T cells are the principal source of interleukin-5 mRNA in allergen-induced allergic rhinitis. Am. J. Respir. Cell Mol. Biol. 9, 356–360.

    CAS  PubMed  Google Scholar 

  18. Ying, S., Durham, S. D., Musuyama, K., Jacobson, M. R., Kay, A. B., and Hamid, Q. (1994) T cells are the principal source of interleukin-4 messenger RNA in the nasal mucosa in allergen-induced rhinitis. Immunol. 82, 200–206.

    CAS  Google Scholar 

  19. Ying, S., Durham, S., Corrigan, C. J., Hamid, Q. and Kay, A. B. (1995) Phenotype of cells expressing mRNA for TH2-type (IL-4 and IL-5) and TH1-type (IL-2 and IFN-γ) cytokines in bronchoalveolar lavage and bronchial biopsies from atopic asthmatics and normal control subjects. Am. J. Respir. CellMol. Biol. 12, 477–478

    CAS  Google Scholar 

  20. Ying, S., Taborada-Barata, L., Meng, Q., Humbert, M., and Kay, A. B. (1995) The kinetics of allergen-induced transcription of messenger RNA for monocyte chemotactic protein-3 (MCP-3) and RANTES in the skin of human atopic subjects: Relationship to eosinophil, T cell, and macrophage recruitment. J. Exp. Med. 181, 2153–2159.

    Article  CAS  PubMed  Google Scholar 

  21. Ying, S, Meng, Q., Barata, L. T., Corrigan, C. J., Barkans, J., Assoufi, B., et al. (1996) Human eosinophils express messenger RNA encoding RANTES and store and release biologically active RANTES protein. Eur. J. Immunol. 26, 70–76

    Article  CAS  PubMed  Google Scholar 

  22. Ying, S., Robinson, D. S., Meng, Q., Rottman, J., Kennedy, R., Ringler, D. J., et al. (1997) Enhanced expression of eotaxin and CCR3 mRNA and protein in atopic asthma and their association with airway hyperresponsiveness and predominant co-localization of eotaxin mRNA to bronchial epithelial and endothelial cells. Eur. J. Immunol. 27, 3507–3516.

    Article  CAS  PubMed  Google Scholar 

  23. Ying, S., Meng, Q., Barata, L. T., Robinson, D. S., Durham, S. R., and Kay. A. B. (1997) Associations between IL-13 and IL-4 (mRNA and protein), VCAM-1 expression and infiltration of eosinophils, macrophages and T cells in the allergen-induced late-phase cutaneous reactions in atopic subjects. J. Immunol. 158, 5050–5057.

    CAS  PubMed  Google Scholar 

  24. Ying, S., Humbert, M., Barkans, J., Corrigan, C. J., Pfister, R., Menz, G., et al. (1997) Expression of IL-4 and IL-5 mRNA and protein product by CD4+and CD8+T cells, eosinophils and mast cells in bronchial biopsies obtained from atopic and non-atopic (intrinsic) asthmatics. J. Immunol. 158, 3539–3544.

    CAS  PubMed  Google Scholar 

  25. Ying, S., Barata, L. T., Meng, Q., Grant, J. A., Barkans, J., Durham, S. R., and Kay, A. B. (1998) High affinity IgE receptor (FceRI)-bearing eosinophils, mast cells, macrophages and Langerhans cells in allergen-induced late-phase cutaneous reactions in atopic subjects. Immunol. 93, 281–288.

    Article  CAS  Google Scholar 

  26. Ying, S., Robinson, D. S, Meng, Q, Barata, L. T., McEuen, A. R., Buckley, M. G., et al. (1999) C-C chemokines in allergen-induced late-phase cutaneous responses in atopic subjects: Association of eotaxin with early 6-hour eosinophils, and eotaxin-2 and MCP-4 with the later 24-hour tissue eosinophilia, and relationship to basophils and other C-C chemokines (MCP-3 and RANTES). J. Immunol. 163, 3976–3984.

    CAS  PubMed  Google Scholar 

  27. Ying, S., Meng, Q., Zeibecoglou, K., Robinson, D. S., Macfarlane, A., Humbert, M., and Kay, A. B. (1999) Eosinophil chemotactic chemokines (eotaxin, eotaxin-2, RANTES, MCP-3, and MCP-4) and CCR3 expression in bronchial biopsies from atopic and non-atopic (intrinsic) asthma. J. Immunol. 1999

    Google Scholar 

  28. Nunez, D. J., Davenport, A. P., Emson, P. C., and Brown, M. J. (1989) A quantitative’ in-situ’ hybridization method using computer-assisted image analysis. Bioch. J. 263, 121–127.

    CAS  Google Scholar 

  29. Marfaing-Koka, A., Devergne, O., Gorgone, G., Schall, T. J., Galanaud, P., and Emilie, D. (1995) Regulation of the production of the RANTES chemokine by endothelial cells. Synergistic induction by IFN-gamma plus TNF-alpha and inhibition by IL-4 and IL-13.J Immunol. 154, 1870–1878.

    CAS  PubMed  Google Scholar 

  30. Wilcox, J. N. (1993) Fundamental principle of in situ hybridization. J. Histochem. Cytochem. 41, 1725–1733.

    CAS  PubMed  Google Scholar 

  31. Henderson, C. (1989) Aminoalkylisane: an inexpensive, simple preparation for slide adhesion. J. Histochnol. 12, 123–124.

    Google Scholar 

  32. Hofler, H, Mueller, J., and Werner, M. (1998) Principle of in situ hybridization, in In Situ Hybridization (Ploak, J. M. and McGee, J. O’D. eds.) Oxford, University Press pp. 1–21.

    Google Scholar 

  33. Coghlam, J. P., Aldred, P., Haralambidis, J., Niall, H. D., Penschow, J. D., and Tregear, G. W. (1985) Hybridization histochemistry. Anal. Biochem. 149, 1–28.

    Article  Google Scholar 

  34. Potts, J. D., Vincent, E. B., Runyan, R. B., and Weeks, D. L. (1992) Sense and antisense TGF-β 3 mRNA levels correlate with cardiac valve induction. Dev. Dynamics 193, 340–345.

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Allergy and Clinical Immunology, National Heart and Lung Institute, Imperial College School of Science, Technology and Medicine, London, UK

    Sun Ying

  2. Allergy and Clinical Immunology, National Heart and Lung Institute, Imperial College School of Science, Technology and Medicine, London, UK

    A. Barry Kay

Authors
  1. Sun Ying
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. Barry Kay
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. National Heart and Lung Institute, Imperial College, London, UK

    Duncan F. Rogers  & Louise E. Donnelly  & 

Rights and permissions

Reprints and permissions

Copyright information

© 2001 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Ying, S., Barry Kay, A. (2001). The Technique of In Situ Hybridization. In: Rogers, D.F., Donnelly, L.E. (eds) Human Airway Inflammation. Methods in Molecular Medicine, vol 56. Humana Press. https://doi.org/10.1385/1-59259-151-5:263

Download citation

  • DOI: https://doi.org/10.1385/1-59259-151-5:263

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-923-0

  • Online ISBN: 978-1-59259-151-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation