Data associated with "Improved methods for single-molecule fluorescence in situ hybridization and immunofluorescence in Caenorhabditis elegans embryos"

This repository contains the data set associated with the Current Protocols publication "Improved methods for single-molecule fluorescence in situ hybridization and immunofluorescence in Caenorhabditis elegans embryos." This data includes the raw images associated with each figure, the complete data sets for both antifade and buffer quantification, and sample output data from both quantification pipelines. In addition, the associated README file describes each file in more detail, including which embryos from the raw data were analyzed and how files were generated.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) visualize the abundance and localization of mRNAs and proteins, respectively, allowing researchers to elucidate the localization, dynamics, and functions of the corresponding genes. Whereas both smFISH and immunofluorescence have been foundational techniques in molecular biology, each protocol has challenges in the C. elegans embryo. smFISH protocols suffer from high initial costs and can photobleach rapidly. Immunofluorescence requires technically challenging permeabilization 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 present protocols to perform smFISH or immunofluorescence alone, including several improvements and optimizations to existing approaches. These protocols include 1) improved fixation and permeabilization steps to preserve cellular morphology while maintaining probe and antibody accessibility in the embryo, 2) a streamlined, in-tube approach for antibody staining that negates freeze-cracking, 3) a previously validated protocol to perform the cost-reducing smiFISH (single molecule inexpensive FISH) adaptation, 4) slide preparation using empirically determined optimal antifade products, and 5) straightforward quantification and data analysis methods. Figure 1 shows a schematic workflow for each protocol. Finally, we discuss tricks and tips to help the reader optimize and troubleshoot individual steps in each protocol. Together, these protocols and optimizations simplify existing protocols 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.National Institutes of Health R35GM124877 Erin Osborne Nishimura NSF GAUSSI training grant DGE-1450032 Dylan Parker NSF GAUSSI training grant DGE-1450032 Lindsay Winkenbac