O dianisidine

Embryos were fixed at 4 °C using 4% PFA followed by four washes (5 min each) in 1X PBST. Then embryos were dyed using o-dianisidine staining solution (40% anhydrous ethanol, 0.65% hydrogen peroxide, 10 mM sodium acetate, and 0.6 mg/ml o-dianisidine [Sigma]) in the dark for 50 min. After incubation, embryos were washed four times (5 min each) in 1X PBST and then added into the bleach solution (1% potassium hydroxide, 3% hydrogen peroxide) until the pigmentation was removed. Embryos were then washed in 1X PBST and imaged by microscope (ZEISS, Imager.A1).

For histochemical staining of haemoglobin, 72 hpf live embryos were incubated with o-dianisidine staining solution (40% ethanol, 0.01 M sodium acetate, 0.65% H₂O₂, 0.6 mg ml⁻¹ o-dianisidine (Sigma, D-9143) for 15 min. Embryos were then washed with PBS, post-fixed in 4% PFA in PBS overnight at 4 °C and stored in 85% glycerol for microscope analysis.

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.

Sudan black B and O-dianisidine staining were performed as described previously (59 (link), 60 (link)).
For Sudan black staining, embryos at indicated stages were fixed with 4% paraformaldehyde (PFA) at 4 °C overnight. After rinsing in phosphate buffered saline with tween 20 (PBST), the embryos were incubated with Sudan black B (Sigma-Aldrich, 199664) working solution for 30 min at room temperature, washed extensively in 70% ethanol, and then progressively rehydrated to PBST. Finally, the embryos were transferred to 100% glycerol, and imaged by a series zoom stereo microscope (Cnoptec, SZ680) connected with a digital camera.
For O-dianisidine staining, live embryos at indicated stages were incubated with O-dianisidine (Sigma-Aldrich, D1943) staining solution for 15 min in the dark, washed with PBST, fixed in 4% PFA for 30 min. After washed with PBST, stained embryos were mounted in 100% glycerol for imaging.

Embryos were first anesthetized with Tricane (Sigma-Aldrich) and then fixed at room temperature for 2 h using 4% paraformaldehyde (PFA) in PBS. To remove PFA, three washes (5 min each) with PBS were performed. Next, embryos were stained with 0.6 mg/mL o-dianisidine (Sigma, USA) 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 wash times (5 min each) with PBS. Then, embryos were incubated into a bleach solution (1% KOH, 3% H₂O₂) for 20 min to remove pigmentation. Finally, the embryos were washed with PBS and imaged immediately. All the embryos with a defect of erythroid cell were selected by imaging and ultimately its genotype confirmed by sequencing.