Optimizing Hybridization Chain Reaction (HCR) In Situ Hybridization in Astyanax Larvae

Fluorescent in-situ hybridization chain reaction (HCR-RNA FISH) is a technique that allows for the simultaneous labeling of multiple target mRNA molecules, signal quantification, and high-resolution oligonucleotide visualization. While this method has been employed to study several common laboratory model systems, its application to non-traditional models is lacking. With the goals of circumventing this limitation and introducing Hybridization Chain Reaction (v3.0) (HCR), the latest version of a new technique for high resolution in situ hybridization, to the Astyanax mexicanus model, we have optimized a novel protocol for this robust evolutionary system. The optimization of HCR in Astyanax mexicanus strengthens the reproducibility of this staining method across a larger number of model organisms while opening the doors to the molecular physiology and genetic study of an insufficiently explored yet promising biological system.