2 Materials
2.1 Embryo
Embedding
- Zebrafish embryos.
- Embryo medium: 5 mM NaCl, 0.17 mM KCl, 0.33 mM
CaCl 2 , 0.33 mM MgSO 4 in deionized water.
Fig. 1Protocol and analysis pipeline for smFISH in zebrafish.(a) Overview of the smFISH method on sections
of zebrafish embryos. (b) Membrane segmentation and transcript detection pipeline.Scale bar:10μm.
Membrane staining (b 1 ) is used to calculate the probability that pixels belong to membrane (green), or
membrane intersection points (vertices,magenta)(b 2 ). Paths are traced from vertices over the membranes to
generate a cell segmentation (b 3 ). Small errors in the segmentation (asteriskandarrowheadinb 3 ) are
manually corrected to finalize the cell mask (b 4 ). smFISH signal (b 5 ) is used to identify single transcripts
(magenta) and transcription foci (white) and combined with the cell (green) and nuclear (blue) segmentations
(b 6 ). Images are maximum projections of 17 z-slices spaced by 0.3μm
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