Whole-Mount RNA In Situ Hybridization and Immunofluorescence of Xenopus Embryos and Tadpoles.
Whole-Mount RNA In Situ Hybridization and Immunofluorescence of Xenopus Embryos and Tadpoles.
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DOI：
10.1101/pdb.prot105635
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发表时间：
2021-10-01
影响因子：
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通讯作者：
Willsey HR
中科院分区：
文献类型：
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作者：
Willsey HR
非洲爪蟾实验的一个主要优点是能够在整个动物的整个发育过程中原位查询内源蛋白和 RNA 的定位。在这里，我描述了三种不同的染色方法，用于可视化发育中的非洲爪蟾胚胎和蝌蚪中的 mRNA 和蛋白质。第一部分概述了适合所有发育阶段的 mRNA 传统比色染色，第二部分扩展了该协议，用于基于荧光的检测，以实现更高的空间和定量分辨率。最后一部分详细介绍了通过免疫荧光检测蛋白质的方法，该方法针对蝌蚪阶段进行了优化，但广泛适用于其他阶段。最后给出了优化策略。
A major advantage of experimentation in Xenopus is the ability to query the localization of endogenous proteins and RNAs in situ in the entire animal during all of development. Here I describe three variations of stainings to visualize mRNAs and proteins in developing Xenopus embryos and tadpoles. The first section outlines a traditional colorimetric staining for mRNAs that is suitable for all stages of development, and the second extends this protocol for fluorescence-based detection for higher spatial and quantitative resolution. The final section details detection of proteins by immunofluorescence, optimized for tadpole stages but widely applicable to others. Finally, optimization strategies are provided.