Hybridisation chain reaction in situ

For in situ HCR, a kit containing a DNA probe set, a DNA HCR amplifier, and hybridisation, wash and amplification buffers were purchased from Molecular Instruments for each target mRNA. The Sox10 and FoxD3 probes initiate B3 (Alexa-546) and B4 (Alexa-546) amplifiers, respectively. In situ HCR protocol was previously detailed by Choi et al. (2016). The protocol provided by the manufacturer was followed. Briefly embryos were fixed with 4% paraformaldehyde for 1 h at room temperature, dehydrated in methanol and stored at −20°C. Following rehydration, embryos were treated with 10 mg/ml Proteinase K for 2.5 min and post-fixed in 4% paraformaldehyde for 20 min. Embryos were incubated with the probes in hybridisation solution overnight at 37°C and, following appropriate washes, incubated in the hairpin solution in amplification buffer overnight at room temperature protected from light.