Drosophila pp 289-302 | Cite as

Fluorescent In Situ Hybridization Protocols in Drosophila Embryos and Tissues

  • Eric Lécuyer
  • Neela Parthasarathy
  • Henry M. Krause
Part of the Methods in Molecular Biology book series (MIMB, volume 420)

Abstract

Fluorescent in situ hybridization is the standard method for visualizing the spatial distribution of RNA. Although traditional histochemical RNA detection methods suffered from limitations in resolution or sensitivity, the recent development of peroxidase-mediated tyramide signal amplification provides strikingly enhanced sensitivity and subcellular resolution. In this chapter, we describe optimized fluorescent in situ hybridization protocols for Drosophila embryos and tissues utilizing tyramide signal amplification, either for single genes or in a high-throughput format, which greatly increases the sensitivity, consistency, economy, and throughput of the procedure. We also describe variations of the method for RNA-RNA and RNA-protein codetection.

Key Words

Drosophila melanogaster embryos and tissues FISH fluorescent in situ hybridization RNA-protein costaining single and double labeling tyramide signal amplification 

References

  1. 1.
    Paddock, S. W., Langeland, A., DeVries, P. J., and Carroll, S. B. (1993) Three-color immunofluorescence imaging of Drosophila embryos by laser scanning confocal microscopy. BioTechniques 14, 42–47.PubMedGoogle Scholar
  2. 2.
    Tautz, D. and Pfeiffle, 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.CrossRefPubMedGoogle Scholar
  3. 3.
    Hughes, S. and Krause, H. M. (1998) Double labeling with FISH in Drosophila whole-mount embryos. BioTechniques 24, 530–532.PubMedGoogle Scholar
  4. 4.
    Hughes, S. and Krause, H. M. (1998) Single and double FISH protocols for Drosophila, in Protocols in confocal microscopy, (Paddock, S., ed.), Humana, Totowa, NJ, pp. 93–101.CrossRefGoogle Scholar
  5. 5.
    Raap, A. K., van de Corput, M. P., Vervenne, R. A., et al. (1995) Ultra-sensitive FISH using peroxidase-mediated deposition of biotin-or fluorochrome tyramides. Human Mol. Genetics 4, 529–534.CrossRefGoogle Scholar
  6. 6.
    Wilkie, G. S. and Davis, I. (1998) Visualizing mRNA by in situ hybridization using high resolution and sensitive tyramide signal amplification. Elsevier Trends J., Technical Tips Online T01458.Google Scholar
  7. 7.
    Kosman, D., Mizutani, C. M., Lemons, D., Cox, W. G., McGinnis, W., and Bier, E. (2004) Multiplex detection of RNA expression in Drosophila embryos. Science 305, 846.CrossRefPubMedGoogle Scholar
  8. 8.
    Tomancak, P., Beaton, A., Weiszmann, R., et al. (2002) Systematic determination of patterns of gene expression during Drosophila embryogenesis. Genome Biol. 3, Research 0088.1–0088.14.Google Scholar
  9. 9.
    Speel, E. J., Ramaekers, F. C., and Hopman, A. H. (1997) Sensitive multicolor fluorescence in situ hybridization using catalyzed reporter deposition (CARD) amplification. J. Histochem. Cytochem. 45, 1439–1446.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2008

Authors and Affiliations

  • Eric Lécuyer
    • 1
  • Neela Parthasarathy
    • 1
  • Henry M. Krause
    • 1
  1. 1.Donnelly Centre for Cellular and Biomolecular ResearchUniversity of TorontoTorontoCanada

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