Single-Molecule Fluorescent In Situ Hybridization (smFISH) on Whole-Mount Planarians

Abstract

Since the establishment of planarian species as laboratory models, investigation of molecular pathways has relied heavily on visualization of transcripts using in situ hybridization (ISH). ISH has revealed various aspects ranging from anatomical details of different organs to distribution of planarian stem cell populations and signaling pathways involved in their unique regenerative response. High-throughput sequencing techniques including single-cell approaches have allowed us to investigate gene expression and cell lineages in more detail. One application that could provide important new insights into more subtle intercellular transcriptional differences and intracellular mRNA localization is single-molecule fluorescent in situ hybridization (smFISH). In addition to obtaining an overview of the expression pattern, this technique allows for single-molecule resolution and hence quantification of a transcript population. This is achieved by hybridization of individual oligonucleotides antisense to a transcript of interest, all carrying a single fluorescent label. This way, a signal is produced only when the combination of labelled oligonucleotides, targeting the same transcript, are hybridized, minimizing background and off-target effects. Moreover, it requires only a few steps compared to the conventional ISH protocol and thus saves time. Here we describe a protocol for the tissue preparation, probe synthesis, and smFISH, combined with immunohistochemistry, for whole-mount Schmidtea mediterranea samples.