Microinjection of β-glucan into the circulation of zebrafish embryos. (A) Photo showing injection setup. (B) Photo showing embryos arrayed in a Petri dish with microinjection needle in position. (C) Image of 0.01 mm microscope stage slide micrometre. The black circle indicates the size of injection bolus that will deliver 1 nL of solution. (D) Image of 2.25 dpf (54 hpf) embryos arrayed in 3% methylcellulose. The loaded microinjection needle approaches from the right to inject embryos sequentially. (E) Image shows individual embryo with needle positioned for microinjection. The white arrowhead indicates the tip of the needle in the embryo at the correct injection position. Black dashed box is magnified view. In magnified view, the black asterisk indicates the position where the needle enters the yolk sac prior to the injection site. The white arrowhead indicates where the needle can be seen to press against the pericardial wall, which means the needle is deep enough to inject into the circulation. (E′) Image shows the same embryo in (E) immediately following microinjection. The white arrowhead indicates the tip of the needle in the embryo. The black asterisk highlights the red colour of the phenol red dye, indicating successful injection. Black dashed box is magnified view. Scale bars 500 μm in (D), 50 μm in (E, E′), and 40 μm in (E, E′ magnified views).

Gene expression analysis following β-glucan injection. (A) Schematic illustrating the injection protocol followed by time points of sample collection for subsequent RT qPCR. (B) Fold change of relevant immune genes at 1, 4, and 11 days post injection (dpi) with β-glucan relative to control. Data shown is from averaged ΔCt values from three biological replicates, n = 15 larvae per biological replicate. Endogenous control gene was ef1a.