The protocol involves investigating the spatiotemporal localization of sox2 and tbxta mRNA transcripts in zebrafish embryos across six developmental stages using RNA in situ hybridization and imaging. Thirty embryos were collected at each stage, dechorionated, fixed, and subjected to RNA in situ hybridization using a probe set, followed by incubation with DAPI and whole-mounting in 80% glycerol before imaging with a confocal microscope.
- Zebrafish embryos at 10, 12, 14, 16, 19 hpf, and 1dpf developmental stages (30 embryos per stage)
- Pronase solution (1 mg/ml)
- Egg water
- 4% paraformaldehyde solution
- Probe set for sox2 and tbxta mRNA transcripts (obtained from Molecular Instruments)
- DAPI solution (1:3000 dilution)
- 80% glycerol solution
- Collect 30 zebrafish embryos at each of the six developmental stages (10, 12, 14, 16, 19 hpf, and 1dpf).
- Dechorionate the embryos using a 1 mg/ml pronase solution for no longer than 90 seconds.
- Wash the embryos thoroughly with egg water.
- Fix the embryos with 4% paraformaldehyde solution overnight at 4°C.
- Perform RNA in situ hybridization according to the Molecular Instruments RNA FISH protocol for zebrafish embryos (MI-Protocol-RNAFISH-Zebrafish-Rev9, adapted from Choi et al.54) using the probe set.
- Incubate the embryos with DAPI (1:3000 dilution) for 20 minutes during the first wash step.
- Gradually transfer the embryos to an 80% glycerol solution for storage.
- Carefully whole mount the embryos in 80% glycerol on a glass slide.
- Image the embryos using a confocal microscope (e.g. Andor Dragonfly).