Skip to main content

Multicolor Whole-Mount In Situ Hybridization

  • Protocol
Developmental Biology Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 137))

Abstract

In situ hybridization is a procedure that allows the detection of the site(s) of transcription of a given gene at a cellular level within an entire organism. Since its introduction into developmental biology (1), this procedure has become an indispensable tool to investigate gene expression; initial protocols employed tritiated probes. These procedures required very long exposure times and allowed the detection of transcripts only on tissue sections. The method was drastically improved by the use of nonradioactive digoxigenin-labeled probes. This allowed the detection of gene transcription also in whole-mounted Drosophila embryos (2). This enhanced technique allowed to obtain results within a few days instead of weeks. The Drosophila protocol was quickly adapted to embryos of vertebrate model organisms that are widely used in developmental biology like Xenopus (3), zebrafish (4) and mice (5).

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
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. Hafen, E., Levine, M., Garber, R. L., and Gehring, W. J. (1983) An improved in situ hybridization method for the detection of cellular RNAs in Drosophila tissue sections and its application for localizing transcripts of the homeotic Antennapedia gene complex. EMBO J. 2, 617–623.

    PubMed  CAS  Google Scholar 

  2. Tautz, D. and Pfeifle, C. (1989) A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81–85.

    Article  PubMed  CAS  Google Scholar 

  3. Hemmati-Brivanlou, A., Frank, D., Bolce, M. E., Brown, B. D., Sive, H. L., and Harland, R. M. (1990) Localization of specific mRNAs in Xenopus embryos by whole-mount in situ hybridization. Development 110, 325–330.

    PubMed  CAS  Google Scholar 

  4. Schulte-Merker, S., Ho, R. K., Herrmann, B. G., and Nüsslein-Volhard, C. (1992) The protein product of the zebrafish homologue of the mouse T-gene is expressed in nuclei of the germ ring and the notochord of the early embryo. Development 116, 1021–1032.

    PubMed  CAS  Google Scholar 

  5. Rosen, B. and Beddington, R. S. P. (1993) Whole-mount in situ hybridization in the mouse embryo: gene expression in three dimensions. Trends Genet 9, 162–167.

    Article  PubMed  CAS  Google Scholar 

  6. Hauptmann, G. and Gerster, T. (1994) Two color whole-mount in situ hybridization to vertebrate and Drosophila embryos. Trends Genet 10, 266.

    Article  PubMed  CAS  Google Scholar 

  7. Hauptmann, G. and Gerster, T. (1996) Multicolor whole-mount in situ hybridization to Drosophila embryos. Dev. Genes Evol. 206, 292–295.

    Article  CAS  Google Scholar 

  8. Jowett, T. and Lettice, L. (1994) Whole-mount in situ hybridizations on zebrafish embryos using a mixture of digoxigenin-and fluorescein-labelled probes. Trends Genet 10, 73–74.

    Article  PubMed  CAS  Google Scholar 

  9. Grossniklaus, U., Pearson, R. K., and Gehring, W. J. (1992) The Drosophila sloppy paired locus encodes two proteins involved in segmentation that show homology to mammalian transcription factors. Genes Dev. 6, 1030–1051.

    Article  PubMed  CAS  Google Scholar 

  10. Walldorf, U. and Gehring, W. J. (1992) Empty spiracles, a gap gene containing a homeobox involved in Drosophila head development. Embo J. 11, 2247–2259.

    PubMed  CAS  Google Scholar 

  11. Hafen, E., Kuroiwa, A., and Gehring, W. J. (1984) Spatial distribution of transcripts from the segmentation gene fushi tarazu during Drosophila embryonic development. Cell 37, 833–841.

    Article  PubMed  CAS  Google Scholar 

  12. Puschel, A. W., Gruss, P., and Westerfield, M. (1992) Sequence and expression pattern of pax-6 are highly conserved between zebrafish and mice. Development 114, 643–651.

    PubMed  CAS  Google Scholar 

  13. Krauss, S., Johansen, T., Korzh, V., Moens, U., Ericson, J. U., and Fjose, A. (1991) Zebrafish pax[zf-a]: a paired box-containing gene expressed in the neural tube. EMBO J. 10, 3609–3619.

    PubMed  CAS  Google Scholar 

  14. Akimenko, M.-A., Ekker, M., Wegner, J., Lin, W., and Westerfield, M. (1994) Combinatorial expression of three zebrafish genes related to distal-less: Part of a homeobox gene code for the head. J. Neurosci. 14, 3475–3486.

    PubMed  CAS  Google Scholar 

  15. Ekker, M., Wegner, J., Akimenko, M. A., and Westerfield, M. (1992) Coordinate Embryonic Expression of 3 Zebrafish engrailed Genes. Development 116, 1001–1010.

    PubMed  CAS  Google Scholar 

  16. Patel, N. H., Martin-Blanco, E., Coleman, K. G., Poole, S. J., Ellis, M. C., Kornberg, T. B., and Goodman, C. S. (1989) Expression of engrailed proteins in arthropods, annelids and chordates. Cell 58, 955–968.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2000 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Hauptmann, G., Gerster, T. (2000). Multicolor Whole-Mount In Situ Hybridization. In: Tuan, R.S., Lo, C.W. (eds) Developmental Biology Protocols. Methods in Molecular Biology™, vol 137. Humana Press. https://doi.org/10.1385/1-59259-066-7:139

Download citation

  • DOI: https://doi.org/10.1385/1-59259-066-7:139

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-854-7

  • Online ISBN: 978-1-59259-066-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics