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Open Access Highly Accessed Methodology article

In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea

Ryan S King and Phillip A Newmark*

Author Affiliations

Howard Hughes Medical Institute, Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, 601 South Goodwin Avenue, Urbana, IL 61801, USA

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BMC Developmental Biology 2013, 13:8  doi:10.1186/1471-213X-13-8

Published: 12 March 2013

Abstract

Background

The freshwater planarian Schmidtea mediterranea has emerged as a powerful model for studies of regenerative, stem cell, and germ cell biology. Whole-mount in situ hybridization (WISH) and whole-mount fluorescent in situ hybridization (FISH) are critical methods for determining gene expression patterns in planarians. While expression patterns for a number of genes have been elucidated using established protocols, determining the expression patterns for particularly low-abundance transcripts remains a challenge.

Results

We show here that a short bleaching step in formamide dramatically enhances signal intensity of WISH and FISH. To further improve signal sensitivity we optimized blocking conditions for multiple anti-hapten antibodies, developed a copper sulfate quenching step that virtually eliminates autofluorescence, and enhanced signal intensity through iterative rounds of tyramide signal amplification. For FISH on regenerating planarians, we employed a heat-induced antigen retrieval step that provides a better balance between permeabilization of mature tissues and preservation of regenerating tissues. We also show that azide most effectively quenches peroxidase activity between rounds of development for multicolor FISH experiments. Finally, we apply these modifications to elucidate the expression patterns of a few low-abundance transcripts.

Conclusion

The modifications we present here provide significant improvements in signal intensity and signal sensitivity for WISH and FISH in planarians. Additionally, these modifications might be of widespread utility for whole-mount FISH in other model organisms.

Keywords:
Planarian; Whole-mount in situ hybridization (WISH); Fluorescent in situ hybridization (FISH); Tyramide signal amplification (TSA); Autofluorescence; Multicolor FISH; Peroxidase quenching; Regeneration; Heat-induced antigen retrieval (HIAR)