Zebrafish microinjection

Zebrafish larvae were injected at 2 days post fertilisation (dpf) and monitored until a maximum of 10 dpf. Larvae were anesthetised by immersion in 0.168 mg/mL tricaine in E3 and transferred onto 3% methyl cellulose in E3 for injection. 1nl of the suspension of cryptococcal cells, where 1nl contained 25 cfu, 200 cfu or 1000 cfu, was injected into the yolk sac circulation valley. For micro-injection of GFP fluorescent beads (Fluoresbrite YG Carboxylate Microspheres 4.50μm). The bead stock solution was pelleted at 78g for 3 minutes, and re-suspended in PVP in phenol red as above for the required concentration. Micro-injection of 40kDa FITC-dextran at 3 dpf in a 50:50 dilution in PVP in phenol red, injected 1 nl into the duct of Cuvier. For micro-injection of 40kDa FITC-dextran at 5 dpf, dextran was resuspended in PBS and 1 nl injected into the duct of Cuvier. Larvae were transferred to fresh E3 to recover from anaesthetic. Any zebrafish injured by the needle/micro-injection, or where infection was not visually confirmed with the presence of phenol red, were removed from the procedure. Zebrafish were maintained at 28°C.