Developing a Robust Multiround HCR-FISH Method Modified for Caenorhabditis elegans

Visualizing multiplexed gene expression patterns at cellular resolution is fundamental for understanding complex biological systems. Iterative protocols, such as sequential fluorescent in situ hybridization (FISH) have been widely used for gene expression profiling in complex organs including animal brains. Caenorhabditis elegans is an extensively studied model organism in neuroscience and genetics. It should greatly benefit from sequential FISH protocols. However, no such iterative protocol optimized for whole-body C. elegans samples has been established. In this study, a multiround hybridization chain reaction FISH method optimized for C. elegans was developed. Initially, simple hydrogel embedding was insufficient, causing over 60% sample loss during multiple probing cycles. This limitation was resolved by chemically attaching the hydrogel to a coverglass. This straightforward step markedly improved sample retention to > 99% across multiple probing cycles. This optimized protocol maintains high signal fidelity, achieving > 86% detection efficiency and > 80% spot-matching reproducibility between rounds. As a proof of concept, this method was successfully applied to a single animal immediately after whole-brain neural activity measurements to profile multiple genes. This accessible protocol provides a promising framework toward correlating multiplexed molecular data with previous functional measurements at a cellular resolution in the same C. elegans individual.