# Developing a Robust Multiround HCR‐FISH Method Modified for Caenorhabditis elegans

## TL;DR

Researchers developed a new FISH method for C. elegans that allows high-resolution gene expression profiling after functional imaging.

## Contribution

A multiround HCR-FISH protocol with >99% sample retention and high fidelity for C. elegans.

## Key findings

- Sample retention exceeded 99% after chemically attaching hydrogel to coverglass.
- Signal fidelity achieved >86% detection efficiency and >80% spot-matching reproducibility.
- The method successfully profiled multiple genes in a single C. elegans after whole-brain activity measurements.

## Abstract

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.

In this study, a multiround HCR‐FISH protocol modified for whole‐mounted
C. elegans
samples was developed using a technique that attaches hydrogel‐embedded samples to coverglass. This achieved a sample retention rate exceeding 99% and high signal fidelity, enabling multiplex gene profiling in the same individual after whole‐brain functional imaging.