Whole-mount O-dianisidine Staining of Zebrafish Embryos

o-dianisidine staining was performed as previously described^{23 (link)}.Briefly, embryos at 48 hpf were first anesthetized with Tricane and then fixed with 4% paraformaldehyde (PFA) at room temperature for 2 h. They were then washed three times with PBS (5 min each) to remove PFA. The embryos were then stained with 0.6 mg/mL o-dianisidine (Sigma Aldrich) in an o-dianisidine staining solution (40% ethanol, 0.65% H₂O₂, 10 mmol/L Na-Acetate) for 30 min in dark and followed by four PBS washeses (5 min each). Then, the embryos were incubated into a bleach solution (1% KOH, 3% H₂O₂) for 20 min to remove pigmentation. Images of stained embryos were taken immediately after rinsing with PBS. Finally, genotypes were confirmed in all samples by HTS.