Multiplexed gene expression analysis in dissociated and intact planarian flatworm tissues using in situ hybridization chain reaction

Planarians are an important model system for studying whole-body regeneration. The visualization of gene expression patterns during this process is an essential experimental technique to understand the underlying mechanisms. Multi-colour fluorescent in situ hybridization (FISH) is currently the standard approach for gene expression analysis. However, the small number of available haptens limits probe multiplexing options, and the required sequential development of individual probes can be extremely time consuming. In situ hybridization chain reaction (HCR) is a newer technique that promises parallel hybridization of multiple genes via annealing of fluorescently labelled hairpins to target sequences. While HCR is well established in several model organisms, including mice and Drosophila, existing protocols perform poorly on whole-mount planarians. Here, we report a systematically optimized HCR protocol that is faster and less labor-intensive than traditional FISH. This method can be applied to dissociated planarian cells or combined with the established DEEP Clear method to label whole-mount animals. These improvements establish HCR as a useful addition to the planarian tool kit, particularly for multiplex gene expression analysis.