Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado.
Curr Protoc. 2021 Nov;1(11):e299. doi: 10.1002/cpz1.299.
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 single molecule inexpensive FISH (smiFISH) 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. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sequential immunofluorescence and single-molecule fluorescence in situ hybridization Alternate Protocol: Abbreviated protocol for simultaneous immunofluorescence and single-molecule fluorescence in situ hybridization Basic Protocol 2: Simplified immunofluorescence in C. elegans embryos Basic Protocol 3: Single-molecule fluorescence in situ hybridization or single-molecule inexpensive fluorescence in situ hybridization.
在秀丽隐杆线虫中对基因产物进行可视化，为在其天然环境中许多新基因的分子和生物学功能提供了深入的了解。单分子荧光原位杂交 (smFISH) 和免疫荧光 (IF) 分别能够可视化 mRNA 和蛋白质的丰度和定位，使研究人员能够最终阐明相应基因的定位、动态和功能。虽然 smFISH 和免疫荧光都是分子生物学的基础技术，但每种方案在秀丽隐杆线虫胚胎中的应用都存在挑战。smFISH 方案初始成本高，容易快速光漂白，而免疫荧光需要技术上具有挑战性的通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，这阻止了在同一样本中探索 mRNA 与相关蛋白质之间的关系。在这里，我们描述了在秀丽隐杆线虫胚胎中按顺序或同时进行免疫荧光和 smFISH 的方案。我们还概述了单独进行 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通透化步骤，以在保持胚胎中探针和抗体可及性的同时保留细胞形态，采用简化的、在管内的抗体染色方法避免了冷冻破裂，验证了降低成本的单分子廉价荧光原位杂交 (smiFISH) 适应方法，使用经验确定的最佳防褪色产品进行载玻片制备，以及简单的定量和数据分析方法。最后，我们讨论了帮助读者优化和解决每个方案中各个步骤的技巧和窍门。总之，这些方案简化了单个分子 RNA 和蛋白质检测的现有工作流程。此外，对感兴趣的蛋白质和 RNA 进行同时、高分辨率成像，将允许对蛋白质和 RNA 分布进行分析、量化和比较，从而进一步了解 RNA 与其蛋白质产物或早期发育中的细胞标记物之间的关系。
