Multiplexed Quantitative In Situ Hybridization with Subcellular or Single-Molecule Resolution Within Whole-Mount Vertebrate Embryos: qHCR and dHCR Imaging (v3.0)
Multiplexed Quantitative In Situ Hybridization with Subcellular or Single-Molecule Resolution Within Whole-Mount Vertebrate Embryos: qHCR and dHCR Imaging (v3.0)
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DOI：
10.1007/978-1-0716-0623-0_10
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发表时间：
2020-01-01
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影响因子：
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通讯作者：
Pierce, Niles A.
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文献类型：
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作者：
Choi, Harry M. T.;Schwarzkopf, Maayan;Pierce, Niles A.
原位杂交的基础上的杂交链反应（HCR）的机制，使多路复用的定量mRNA成像的解剖背景下，整装脊椎动物胚胎。第三代原位HCR（v3.0）在整个协议中提供自动背景抑制，大大提高了性能和易用性。原位HCR v3.0支持两种定量成像模式：（1）qHCR成像，用于在解剖学背景下使用亚细胞分辨率进行模拟mRNA相对定量;（2）dHCR成像，用于在解剖学背景下使用单分子分辨率进行数字mRNA绝对定量。在这里，我们提供了qHCR和dHCR成像在整个安装斑马鱼，鸡，小鼠胚胎的协议。
In situ hybridization based on the mechanism of hybridization chain reaction (HCR) enables multiplexed quantitative mRNA imaging in the anatomical context of whole-mount vertebrate embryos. Third-generation in situ HCR (v3.0) provides automatic background suppression throughout the protocol, dramatically enhancing performance and ease of use. In situ HCR v3.0 supports two quantitative imaging modes: (1) qHCR imaging for analog mRNA relative quantitation with subcellular resolution in an anatomical context and (2) dHCR imaging for digital mRNA absolute quantitation with single-molecule resolution in an anatomical context. Here, we provide protocols for qHCR and dHCR imaging in whole-mount zebrafish, chicken, and mouse embryos.