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An optimized procedure for whole-mount in situ hybridization on mouse embryos and embryoid bodies

Nature Protocols volume 3, pages 1194–1201 (2008)Cite this article

Abstract

Determining the precise expression pattern of a gene of interest at various stages of development is essential to understanding its biological function in embryology. This protocol describes a sensitive method for whole-mount in situ hybridization (WISH) to mouse embryos, using cRNA probes. Adaptations are provided that allow the protocol to be applied to embryonic stages ranging from blastocysts to postimplantation stage embryos, and to embryoid bodies. We also describe an in situ method for differential detection of two probes. Probe labeling and dissection and preparation of the embryos can be performed in 2 d. The actual WISH procedure can be completed in another 3 d.

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Figure 1: Schematic outline of the WISH protocol for postimplantation embryos and EBs.
Figure 2: Gel electrophoresis on a 1% agarose TBE gel for several labeled probes (see Step 8).
Figure 3: Gene expression patterns detected with WISH, using digoxigenin-labeled probes and staining with BM Purple.
Figure 4: Gene expression patterns of Cerberus-like (Cer1) and Brachyury (T) in a postimplantation embryo and EBs, detected with the double in situ protocol.

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Acknowledgements

We thank Ann Pauwels and Ellen Geeroms for their help. The laboratory is supported by the Horizontal Action and Concerted Research Action of the Vrije Universiteit Brussel (Grants HOA1 and GOA29), the Flemish Fund for Scientific Research (Grants G.0485.06 and G.0307.04) and the EU (6th Framework, Grant NEST012930). M.H. is an aspirant FWO.

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Author notes

  1. Erik Willems & Caroline R Kemp

    Present address: Present addresses: The Del E. Webb Neuroscience, Aging and Stem Cell Research Center, Burnham Institute for Medical Researchs, 10901 North Torrey Pines Road, La Jolla, California 92037, USA (E.W.).,

  2. Denis Piette, Marijke Hendrickx, Erik Willems, Caroline R Kemp and Luc Leyns: D.P., M.H. and E.W., and C.R.K. and L.L. contributed equally to this work.

  3. Regulatory Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA (C.R.K.).

Authors and Affiliations

  1. Department of Biology, Vrije Universiteit Brussel (VUB), Lab for Cell Genetics, Pleinlaan 2, Brussels, 1050, Belgium

    Denis Piette, Marijke Hendrickx, Erik Willems, Caroline R Kemp & Luc Leyns

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Correspondence to Caroline R Kemp or Luc Leyns.

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Piette, D., Hendrickx, M., Willems, E. et al. An optimized procedure for whole-mount in situ hybridization on mouse embryos and embryoid bodies. Nat Protoc 3, 1194–1201 (2008). https://doi.org/10.1038/nprot.2008.103

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