Formaldehyde-based whole-mount in situ hybridization method for planarians

Authors

  • Bret J. Pearson,

    1. Department of Neurobiology and Anatomy, Howard Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City, Utah
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    • Drs. Pearson, Eisenhoffer, Gurley, and Rink contributed equally to this work.

  • George T. Eisenhoffer,

    1. Department of Neurobiology and Anatomy, Howard Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City, Utah
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    • Drs. Pearson, Eisenhoffer, Gurley, and Rink contributed equally to this work.

  • Kyle A. Gurley,

    1. Department of Neurobiology and Anatomy, Howard Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City, Utah
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    • Drs. Pearson, Eisenhoffer, Gurley, and Rink contributed equally to this work.

  • Jochen C. Rink,

    1. Department of Neurobiology and Anatomy, Howard Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City, Utah
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    • Drs. Pearson, Eisenhoffer, Gurley, and Rink contributed equally to this work.

  • Diane E. Miller,

    1. Department of Neurobiology and Anatomy, Howard Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City, Utah
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  • Alejandro Sánchez Alvarado

    Corresponding author
    1. Department of Neurobiology and Anatomy, Howard Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City, Utah
    • Department of Neurobiology and Anatomy, Howard Hughes Medical Institute, University of Utah School of Medicine, 401 MREB, 20N 1900E, Salt Lake City, UT 84132
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Abstract

Whole-mount in situ hybridization (WISH) is a powerful tool for visualizing gene expression patterns in specific cell and tissue types. Each model organism presents its own unique set of challenges for achieving robust and reproducible staining with cellular resolution. Here, we describe a formaldehyde-based WISH method for the freshwater planarian Schmidtea mediterranea developed by systematically comparing and optimizing techniques for fixation, permeabilization, hybridization, and postprocessing. The new method gives robust, high-resolution labeling in fine anatomical detail, allows co-labeling with fluorescent probes, and is sufficiently sensitive to resolve the expression pattern of a microRNA in planarians. Our WISH methodology not only provides significant advancements over current protocols that make it a valuable asset for the planarian community, but should also find wide applicability in WISH methods used in other systems. Developmental Dynamics 238:443–450, 2009. © 2009 Wiley-Liss, Inc.

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