A safer fluorescent in situ hybridization protocol for cryosections

Fluorescent in situ hybridization (FISH) enables highly sensitive, high-resolution detection of gene transcripts. Moreover, by employing multiple probes, this technique allows multiplexed, simultaneous detection of distinct gene expression patterns in a spatiotemporal manner, making it a valuable spatial transcriptomics approach. Owing to these advantages, FISH techniques are rapidly being adopted across diverse areas of basic biology. However, conventional protocols often rely on volatile, toxic reagents such as formalin or methanol, posing potential health risks to researchers. Here, we present a safer protocol that replaces these chemicals with low-toxicity and commercially available alternatives, without compromising the high detection sensitivity of FISH. We validated this protocol using both in situ hybridization chain reaction (HCR) and signal amplification by exchange reaction (SABER)-FISH in frozen sections of amphibians, Pleurodeles waltl and Xenopus laevis , as well as the teleost medaka ( Oryzias latipes ). Our results demonstrate successful multiplexed detection of various morphogenetic genes in these amphibian models and cell-type marker genes in the medaka using this safer protocol. The protocol has the additional advantage of requiring no proteolytic enzyme treatment. This protocol retains the benefits of high-sensitivity detection afforded by in situ HCR and SABER-FISH while providing a safer option for researchers, thereby offering a valuable tool for both basic biological and medical studies.