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A modified protocol for the detection of three different mRNAs with a new-generation in situ hybridization chain reaction on frozen sections

Sui, QQ; Zhu, J; Li, X; Knight, GE; He, C; Burnstock, G; Yuan, H; (2016) A modified protocol for the detection of three different mRNAs with a new-generation in situ hybridization chain reaction on frozen sections. Journal of Molecular Histology , 47 (6) pp. 511-529. 10.1007/s10735-016-9696-x. Green open access

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Abstract

A new multiple fluorescence in situ hybridization method based on hybridization chain reaction was recently reported, enabling simultaneous mapping of multiple target mRNAs within intact zebrafish and mouse embryos. With this approach, DNA probes complementary to target mRNAs trigger chain reactions in which metastable fluorophore-labeled DNA hairpins self-assemble into fluorescent amplification polymers. The formation of the specific polymers enhances greatly the sensitivity of multiple fluorescence in situ hybridization. In this study we describe the optimal parameters (hybridization chain reaction time and temperature, hairpin and salt concentration) for multiple fluorescence in situ hybridization via amplification of hybridization chain reaction for frozen tissue sections. The combined use of fluorescence in situ hybridization and immunofluorescence, together with other control experiments (sense probe, neutralization and competition, RNase treatment, and anti-sense probe without initiator) confirmed the high specificity of the fluorescence in situ hybridization used in this study. Two sets of three different mRNAs for oxytocin, vasopressin and somatostatin or oxytocin, vasopressin and thyrotropin releasing hormone were successfully visualized via this new method. We believe that this modified protocol for multiple fluorescence in situ hybridization via hybridization chain reaction would allow researchers to visualize multiple target nucleic acids in the future.

Type: Article
Title: A modified protocol for the detection of three different mRNAs with a new-generation in situ hybridization chain reaction on frozen sections
Location: Netherlands
Open access status: An open access version is available from UCL Discovery
DOI: 10.1007/s10735-016-9696-x
Publisher version: http://doi.org/10.1007/s10735-016-9696-x
Language: English
Additional information: This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
Keywords: Brain section, Hybridization chain reaction, Multiple fluorescence in situ hybridization
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Neuro, Physiology and Pharmacology
URI: http://discovery.ucl.ac.uk/id/eprint/1524706
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