RT Journal Article SR Electronic T1 Improved methods for protein and single-molecule RNA detection in C. elegans embryos JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.05.07.443170 DO 10.1101/2021.05.07.443170 A1 Dylan M. Parker A1 Lindsay P. Winkenbach A1 Annemarie Parker A1 Sam Boyson A1 Erin Osborne Nishimura YR 2021 UL http://biorxiv.org/content/early/2021/05/08/2021.05.07.443170.abstract AB 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 many genes. Here, we describe several improvements and optimizations to existing IF and smFISH approaches specifically for use in C. elegans embryos. We present 1) optimized fixation and permeabilization steps to preserve cellular morphology while maintaining probe and antibody accessibility, 2) a streamlined, in-tube approach that negates freeze-cracking, 3) the smiFISH (single molecule inexpensive FISH) adaptation that reduces cost, 4) an assessment of optimal anti-fade products, and 5) straightforward quantification and data analysis methods. Most importantly, published IF and smFISH protocols have predominantly been mutually exclusive, preventing exploration of relationships between an mRNA and a relevant protein in the same sample. Here, we present methods to combine IF and smFISH protocols in C. elegans embryos including an efficient method harnessing nanobodies. Finally, we discuss tricks and tips to help the reader optimize and troubleshoot individual steps in each protocol.Competing Interest StatementThe authors have declared no competing interest.