Peripheral blood cells were collected from euthanized zebrafish by tail amputation in adults and by heart puncture in embryos or larvae. Blood smears were made on superfrost/plus slides (Fisher, Cat.12-550-15) and fixed in 100% methanol for 15 s. To visualize erythrocyte morphology, cells were stained using a Hema3 kit (Fisher Diagnostics, Cat. 123–869, an analog to Wright–Giemsa method). To stain for hemoglobin, cells were incubated with an o-dianisidine staining buffer (0.6 mg/ml o-dianisidine, 0.01 M sodium acetate, pH 4.5, 0.65% H2O2, and 40% ethanol (vol/vol)) for 15 min in the dark. The slides were imaged with a BZ9000 Keyence microscope. For the whole mount o-dianisidine staining, dechorionated and euthanized embryos or larvae were incubated with the o-diansidine staining buffer for 15 min in the dark, and the embryos or larvae were photographed with a Leica CTR5000 microscope. The images shown in Figs. 1 and 2 are representative results from at least two independent experiments, with at least three embryos or larvae in each group per experiment.
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