Optimizing the hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) protocol for Pleurodeles waltl.

Advances in transcriptomic technologies have transformed the study of complex biological processes, including tissue regeneration, through high-resolution characterization of gene expression programs. In regenerative vertebrate models such as the Iberian ribbed newt (Pleurodeles waltl), these approaches provide insight into the molecular mechanisms underlying retina and lens regeneration. However, single-cell RNA sequencing studies lack spatial resolution, meaning the ability to validate gene expression patterns within ocular tissues is essential and requires optimization. In this study, we optimized hybridization chain reaction fluorescent in situ hybridization (HCR-FISH) for use in P. waltl eyes. HCR-FISH enables specific mRNA detection through split-initiator probes and hairpin-based signal amplification with automatic background suppression. Because incomplete genome annotation in emerging model organisms complicates transcript selection and probe design, we optimized an optional in silico workflow for transcript screening, orthology confirmation, and probe generation. We systematically optimized tissue processing parameters to preserve tissue integrity while enhancing signal quality. To overcome imaging constraints from pigmented ocular tissues, we implemented a whole-mount protocol with optional bleaching and cryosectioning, improving visualization without compromising spatial localization. Using this workflow, we detected retinal markers SLC1A3 (Müller glia cells) and RPE65 (retinal pigment epithelium) within the newt eye. Notably, the RPE65 probe was designed in house and showed comparable detection to a Molecular Instruments probe across two sample preparation protocols. This study presents a reproducible framework for spatial transcript detection in emerging eye regenerative models, while integrating transcriptomic and anatomical data. Together, our design-to-detection pipeline strengthens spatial validation of RNA sequencing profiles in P. waltl.