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Sensitive Whole-Mount Fluorescent In Situ Hybridization in Zebrafish Using Enhanced Tyramide Signal Amplification

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Brain Development

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1082))

Abstract

Whole-mount in situ hybridization is the preferred method for detecting transcript distributions in whole embryos, tissues, and organs. We present here a sensitive fluorescent in situ hybridization method for colocalization analysis of different transcripts in whole embryonic zebrafish brains. The method is based on simultaneous hybridization of differently hapten-labeled RNA probes followed by sequential rounds of horseradish peroxidase (POD)-based transcript detection. Sequential detection involves enhancement of fluorescent signals by tyramide signal amplification (TSA) and effective inactivation of the antibody–POD conjugate prior to the following detection round. We provide a detailed description of embryo preparation, hybridization, antibody detection, POD–TSA reaction, and mounting of embryos for imaging. To achieve high signal intensities, we optimized key steps of the method. This includes improvement of embryo permeability by hydrogen peroxide treatment and efficacy of hybridization and TSA–POD reaction by addition of the viscosity-increasing polymer dextran sulfate. The TSA–POD reaction conditions are further optimized by application of substituted phenol compounds as POD accelerators and use of highly efficient bench-made tyramide substrates. The obtained high signal intensities and cellular resolution of our method allows for co-expression analysis and generation of three-dimensional models. Our protocol is tailored to optimally work in zebrafish embryos, but can surely be modified for application in other species as well.

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Acknowledgments

The Stockholm Greater Council and the Karolinska Institutet supported this work. Imaging was in part performed at the Live Cell Imaging Unit, Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden, supported by grants from the Knut and Alice Wallenberg Foundation, the Swedish Research Council, and the Centre for Biosciences.

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Authors and Affiliations

  1. Department of Biosciences and Nutrition, NOVUM, Karolinska Institutet, Huddinge, Sweden

    Gilbert Lauter, Iris Söll & Giselbert Hauptmann

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  1. Gilbert Lauter
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  2. Iris Söll
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  3. Giselbert Hauptmann
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  1. Department of Biology, University of Fribourg, Fribourg, Switzerland

    Simon G Sprecher

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Lauter, G., Söll, I., Hauptmann, G. (2014). Sensitive Whole-Mount Fluorescent In Situ Hybridization in Zebrafish Using Enhanced Tyramide Signal Amplification. In: Sprecher, S. (eds) Brain Development. Methods in Molecular Biology, vol 1082. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-655-9_12

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  • DOI: https://doi.org/10.1007/978-1-62703-655-9_12

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-654-2

  • Online ISBN: 978-1-62703-655-9

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