Multicolored Visualization of Transcript Distributions in Drosophila Embryos

Part of the Neuromethods book series (NM, volume 99)


Despite large-scale gene expression profiling studies, it is still often required to precisely characterize the localization of different transcripts in relation to each other to determine unique and overlapping expression sites at a cellular level. We describe here a versatile protocol for simultaneous examination of three unique mRNA expression patterns in Drosophila melanogaster embryos. Three differently labeled antisense RNA probes are hybridized together to the embryos and detected by sequential alkaline phosphatase-based immunohistochemistry. Transcript distributions are revealed by colorimetric enzymatic reactions that permit to highlight each mRNA expression pattern by a differing and contrasting cellular color precipitate. We provide tips and tricks for each critical step helpful for successful application of the tricolor whole-mount in situ hybridization (WISH) method.

Key words

Digoxigenin Biotin Fluorescein Azo dye Fast Blue Fast Red INT Alkaline phosphatase substrate Gene expression analysis RNA localization 



We thank Prof. Dr. Ulrich Theopold and the Wenner Gren Institute of Stockholm University for their support, while preparing this manuscript. This method was initially developed during G.H.’s stay in the laboratory of Dr. Thomas Gerster at the Biozentrum of the Universität Basel.


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Molecular Biosciences, The Wenner-Gren InstituteStockholm UniversityStockholmSweden

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