The DOI matches its Crossref record; 49 of the 49 references it cites were found in a registry or an index.
In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea
Open accessAbstract
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.
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Cited by
Cell type transcriptome atlas for the planarian Schmidtea mediterranea
Fincher, C. T., Wurtzel, O., de Hoog, T., Kravarik, K. M., & Reddien, P. W. (2018) · Science · doi:10.1126/science.aaq1736 · Cited by 436
Changes in regeneration-responsive enhancers shape regenerative capacities in vertebrates
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Wnt and TGFβ coordinate growth and patterning to regulate size-dependent behaviour
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Spinal V2b neurons reveal a role for ipsilateral inhibition in speed control
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Metadata
- Authors
- King, R. S., & Newmark, P. A.
- Venue
- BMC Developmental Biology
- Published
- 2013-03-12
- Volume
- 13
- Issue
- 1
- Pages
- 8-8
- ISSN
- 1471-213X (ISSN-L)
- OpenAlex
- W2017874888
- Cited by
- 310
- References
- 49
- FWCI
- 6.72
- Type
- journal article
- Language
- en
- Open access
- hybrid
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