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Wholemount In Situ Hybridization to Xenopus Embryos

  • C. Michael Jones
  • James C. Smith
Part of the METHODS IN MOLECULAR BIOLOGY™ book series (MIMB, volume 461)

1. Introduction

Whole-mount in situ hybridization was first used to detect gene expression in Drosophila embryos (1). Various methods are now used to localize mRNAs in most species used for biological studies, and the methods have proven particularly useful when applied to vertebrate species. Here we provide a protocol commonly used for localizing transcripts in Xenopus embryos. The method is applicable to whole embryos and intact tissue explants. The methods are essentially as originally described by Hemmati-Brivanlou et al. (2) and then modified by Harland (3).

Standard molecular biology techniques not described in detail here can be found in Sambrook et al. (4).

2. Materials

Other reagents and optional equipment are listed in the text where appropriate. Working concentrations and storage conditions for stock solutions are also detailed.

Digoxygenin-11-UTP (Boehringer-Mannheim 1209 256)

Torula RNA

 

Ribonucleotides

Heparin

 

1 M DTT

Tween-20

 

RNasin

CHAPS

 

RNA Polymerase (SP6,T3,...

Keywords

Maleic Acid Acetic Anhydride Hybridization Buffer Xenopus Embryo Acetone Powder 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    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.CrossRefPubMedGoogle Scholar
  2. 2.
    Hemmati-Brivanlou, A., Frank, D., Bolce, M. B., 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.PubMedGoogle Scholar
  3. 3.
    Harland, R. M. (1991) In situ hybridization: an improved whole-mount method for Xenopus embryos, in: Methods in Cell Biology-Xenopus laevis: Practical Uses in Cell and Molecular Biology (Kay, B. K. and Peng, H. B., eds.), Academic, San Diego, CA, pp. 685–695.CrossRefGoogle Scholar
  4. 4.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  5. 5.
    Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K, and Green, M. R. (1985) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12, 7035–7056.CrossRefGoogle Scholar
  6. 6.
    Hayashi, S., Gillam, I. C., Delaney, A., and Tener, G. M. (1978) Acetylation of chromosome squashes of Drosophila melanogaster decreases the background in autoradiographs from hybridization with 125I-labelled RNA. J. Histochem. Cyto-chem. 36, 677–679.Google Scholar
  7. 7.
    Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar

Copyright information

© Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • C. Michael Jones
    • 1
  • James C. Smith
    • 2
  1. 1.Centre for Molecular MedicineSingapore
  2. 2.CRC/Wellcome Trust Gurdon InstituteUniversity of CambridgeCambridgeUK

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