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

Area of Science:

• Molecular Biology
• Developmental Biology
• Regenerative Medicine

Background:

• Planarian flatworms are established model organisms for studying regeneration.
• In situ hybridization (ISH) is a key technique for visualizing transcript distribution in planarians.
• High-throughput sequencing and single-cell approaches have advanced gene expression analysis.

Purpose of the Study:

• To introduce and detail a protocol for single-molecule fluorescent in situ hybridization (smFISH) in planarians.
• To enable visualization of subtle intercellular transcriptional differences and intracellular mRNA localization.
• To provide a more precise quantification of transcript populations compared to conventional ISH.

Main Methods:

• Development of a protocol for tissue preparation suitable for smFISH.
• Synthesis of fluorescently labeled oligonucleotide probes targeting specific transcripts.
• Combination of smFISH with immunohistochemistry for whole-mount *Schmidtea mediterranea* samples.

Main Results:

• The described smFISH protocol allows for single-molecule resolution of transcript detection.
• The technique minimizes background and off-target effects through specific oligonucleotide hybridization.
• The protocol is streamlined, requiring fewer steps than conventional ISH, saving time.

Conclusions:

• smFISH is a valuable technique for detailed analysis of gene expression patterns in planarians.
• This method enhances the study of planarian regeneration by providing single-molecule resolution.
• The protocol facilitates deeper insights into cellular and molecular mechanisms underlying planarian regeneration.