Improved Methods for Single-Molecule Fluorescence In Situ Hybridization and Immunofluorescence in Caenorhabditis elegans Embryos.
Improved Methods for Single-Molecule Fluorescence In Situ Hybridization and Immunofluorescence in Caenorhabditis elegans Embryos.
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DOI：
10.1002/cpz1.299
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发表时间：
2021-11
期刊：
影响因子：
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通讯作者：
Nishimura, Erin Osborne
中科院分区：
文献类型：
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作者：
Parker, Dylan M;Winkenbach, Lindsay P;Parker, Annemarie;Boyson, Sam;Nishimura, Erin Osborne
秀丽隐杆线虫基因产物的可视化为许多新基因在其原生环境中的分子和生物学功能提供了见解。单分子荧光原位杂交（smFISH）和免疫荧光（IF）分别使mRNA和蛋白质的丰度和定位可视化，使研究人员能够最终阐明相应基因的定位，动力学和功能。尽管smFISH和免疫荧光都是分子生物学的基础技术，但每种方案都对C.线虫胚胎smFISH方案的初始成本高，并且可以快速光漂白，而免疫荧光需要技术上具有挑战性的透化步骤和载玻片制备。最重要的是，已发表的smFISH和IF方案主要是相互排斥的，阻止了对同一样品中mRNA和相关蛋白质之间关系的探索。在这里，我们描述的协议进行免疫荧光和smFISH在C。线虫胚胎的顺序或同时。我们还概述了单独执行smFISH或免疫荧光的步骤，包括对现有方法的几项改进和优化。这些方案的特点是改进了固定和透化步骤，以保持细胞形态，同时保持探针和抗体在胚胎中的可及性，这是一种简化的抗体染色管内方法，可消除冷冻裂解，是一种经验证的降低成本的smiFISH方法。（单分子廉价FISH）适应，使用经验确定的最佳抗褪色产品制备载玻片，以及直接的量化和数据分析方法。最后，我们讨论技巧和提示，以帮助读者优化和故障排除每个协议中的各个步骤。总之，这些协议简化了单分子RNA和蛋白质检测的现有工作流程。此外，同时，高分辨率成像的蛋白质和RNA的利益将允许分析，定量和比较蛋白质和RNA的分布，进一步了解RNA和它们的蛋白质产物或细胞标记物之间的关系在早期发展。
Visualization of gene products in Caenorhabditis elegans has provided insights into the molecular and biological functions of many novel genes in their native contexts. Single-molecule Fluorescence In Situ Hybridization (smFISH) and Immunofluorescence (IF) enable the visualization of the abundance and localization of mRNAs and proteins, respectively, allowing researchers to ultimately elucidate the localization, dynamics, and functions of the corresponding genes. Whereas both smFISH and immunofluorescence have been foundational techniques in molecular biology, each protocol poses challenges for use in the C. elegans embryo. smFISH protocols suffer from high initial costs and can photobleach rapidly, and immunofluorescence requires technically challenging permeabilization steps and slide preparation. Most importantly, published smFISH and IF protocols have predominantly been mutually exclusive, preventing the exploration of relationships between an mRNA and a relevant protein in the same sample. Here, we describe protocols to perform immunofluorescence and smFISH in C. elegans embryos either in sequence or simultaneously. We also outline the steps to perform smFISH or immunofluorescence alone, including several improvements and optimizations to existing approaches. These protocols feature improved fixation and permeabilization steps to preserve cellular morphology while maintaining probe and antibody accessibility in the embryo, a streamlined, in-tube approach for antibody staining that negates freeze-cracking, a validated method to perform the cost-reducing smiFISH (single molecule inexpensive FISH) adaptation, slide preparation using empirically determined optimal antifade products, and straightforward quantification and data analysis methods. Finally, we discuss tricks and tips to help the reader optimize and troubleshoot individual steps in each protocol. Together, these protocols simplify existing workflows for single-molecule RNA and protein detection. Moreover, simultaneous, high-resolution imaging of proteins and RNAs of interest will permit analysis, quantification, and comparison of protein and RNA distributions, furthering our understanding of the relationship between RNAs and their protein products or cellular markers in early development.