A zWEDGI Technique to Visualize Fungal Pathogen-Infected Zebrafish Larvae

On the day of imaging, prepare one 3.5-millimeter petri dish with 100 micromolar PTU and one with E3-tricaine. Add E3-tricaine into the chambers of a zWEDGI device, and use a P100 micropipette to remove air bubbles from the chambers and the restraining channel, then, remove all excess E3-tricaine outside of the chambers.

Pipette up one larva and transfer it to the dish with the E3 PTU, then, transfer it to the E3-tricaine with as little liquid as possible. After 30 seconds, transfer the anesthetized larvae into the loading chamber of the wounding and entrapment device.

Remove E3-tricaine from the wounding chamber, and release it into the loading chamber in order to move the tail of the larvae into the restriction channel. Make sure that the larva is positioned on its lateral, dorsal, or dorsolateral side so that the hindbrain can be imaged with an inverted objective lens.

After imaging the larva with a confocal microscope, release E3-tricaine into the wounding chamber to push the larva from the restraining channel into the loading chamber. Pick up the larva and transfer it back to the dish with E3-tricaine, then, transfer it to the dish with E3 PTU with as little liquid as possible. Rinse it in PTU and transfer it back into the 48-well plate.