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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

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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.

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Acknowledgments

This work was supported by the National Natural Science Foundation of the People’s Republic of China (81471260 to Z. Xiang).

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    Authors and Affiliations

    1. Department of Neurobiology, Key Laboratory of Molecular Neurobiology, Ministry of Education, Second Military Medical University, Shanghai, 200433, People’s Republic of China

      Qian-qian Sui, Jiao Zhu, Cheng He & Zhenghua Xiang

    2. Department of Anesthesiology, Changzheng Hospital, Second Military Medical University, Shanghai, 200433, People’s Republic of China

      Xiangnan Li & Hongbin Yuan

    3. Department of Anesthesiology, The Third People’s Hospital of Yancheng, Yancheng, 224001, People’s Republic of China

      Xiangnan Li

    4. Autonomic Neuroscience Centre, University College Medical School, Rowland Hill Street, London, NW3 2PF, UK

      Gillian E. Knight & Geoffrey Burnstock

    5. Department of Pharmacology and Therapeutics, The University of Melbourne, Parkville, VIC, Australia

      Geoffrey Burnstock

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    1. Qian-qian Sui
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    Correspondence to Zhenghua Xiang.

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    The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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    Qian-qian Sui, Jiao Zhu and Xiangnan Li contributed equally to this work.

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    Fig. 1

    VP probe control experiments in the SON of the rat hypothalamus. A, no ISH signals were detected after RNase treatment; B, no ISH signals were detected in the section hybridized with VP anti-sense probes with no initiator; C, no ISH signals were detected in the sections hybridized with VP sense-probes. D, E, F, G and H show the results when the sections were hybridized with different ratios of sense and anti-sense VP probe concentrations (μm). No signals were detected when the ratios were 8:1 or 4:1. Positive signals increased as the ratio decreased from 2:1 to 0:1. All scale bars = 80 μm. (JPEG 1378 kb)

    Fig. 2

    OT probe control experiments in the SON of the rat hypothalamus. A, no ISH signals were detected after RNase treatment; B, no ISH signals were detected in the section hybridized with OT anti-sense probes with no initiator; C, no ISH signals were detected in the sections hybridized with OT sense-probes. D, E, F, G and H show the results when the sections were hybridized with different ratios of sense and anti-sense OT probe concentrations (μm). No signals were detected when the ratios were 8:1 or 4:1. Positive signals increased as the ratio decreased from 2:1 to 0:1. All sections were counterstained with DAPI (4′,6-diamidino-2-phenylindole dihydrochloride). All scale bars = 80 μm. (JPEG 3194 kb)

    Fig. 3

    TRH probe control experiments in periventricular nucleus of the rat hypothalamus. A, no ISH signals were detected after RNase treatment; B, no ISH signals were detected in the section hybridized with TRH anti-sense probes with no initiator; C, no ISH signals were detected in the sections hybridized with TRH sense-probes. D, E, F, G and H show the results when the sections were hybridized with different ratios of sense and anti-sense TRH probe concentrations (μm). No signals were detected when the ratios were 8:1 or 4:1. Positive signals increased as the ratio decreased from 2:1 to 0:1. All sections were counterstained with DAPI. All scale bars = 80 μm. (JPEG 3155 kb)

    Fig. 4

    SST probe control experiments in periventricular nucleus of the rat hypothalamus. A, no ISH signals were detected after RNase treatment; B, no ISH signals were detected in the section hybridized with SST anti-sense probes with no initiator; C, no ISH signals were detected in the sections hybridized with SST sense-probes. D, E, F, G and H show the results when the sections were hybridized with different ratios of sense and anti-sense SST probe concentrations (μm). No signals were detected when the ratios were 8:1 or 4:1. Positive signals increased as the ratio decreased from 2:1 to 0:1. All sections were counterstained with DAPI. All scale bars = 80 μm. (JPEG 3647 kb)

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    Sui, Qq., Zhu, J., Li, X. et al. A modified protocol for the detection of three different mRNAs with a new-generation in situ hybridization chain reaction on frozen sections. J Mol Hist 47, 511–529 (2016). https://doi.org/10.1007/s10735-016-9696-x

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    • DOI: https://doi.org/10.1007/s10735-016-9696-x

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