Single molecule fluorescence in situ hybridization (smFISH) is a method to visualize single mRNA molecules. When combined with cellular and nuclear segmentation, transcripts can be assigned to different cellular compartments resulting in quantitative information on transcript levels at subcellular resolution. The use of smFISH in zebrafish has been limited by the lack of protocols and an automated image analysis pipeline for samples of multicellular organisms. Here we present a protocol for smFISH on zebrafish cryosections. The protocol includes a method to obtain high-quality sections of zebrafish embryos, an smFISH protocol optimized for zebrafish cryosections, and a user-friendly, automated analysis pipeline for cell segmentation and transcript detection. The software is freely available and can be used to analyze sections of any multicellular organism.
Keywords: Automated cell segmentation; Cryosections; Transcript detection; Zebrafish; smFISH.