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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- 作者：Qian-qian Sui ; Jiao Zhu ; Xiangnan Li ; Gillian E. Knight…
- 关键词：Multiple fluorescence in situ hybridization ; Hybridization chain reaction ; Brain section
- 刊名：Journal of Molecular Histology
- 出版年：2016
- 出版时间：December 2016
- 年：2016
- 卷：47
- 期：6
- 页码：511-529
- 全文大小：3,232 KB
- 刊物类别：Biomedical and Life Sciences
- 刊物主题：Life Sciences
Cell Biology
Biomedicine
Developmental Biology - 出版者：Springer Netherlands
- ISSN：1567-2387
- 卷排序：47
文摘
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.