This study utilizes transgenic zebrafish larvae to investigate immune cell dynamics during fungal infection. The larvae are engineered to express fluorescent markers, allowing for real-time tracking of immune responses in the hindbrain.
Begin with transgenic zebrafish larvae expressing fluorescent-labeled immune cells tagged with unique fluorescent markers for precise tracking.
Larvae are pre-injected with red fluorescent protein expressing Aspergillus spores, in their hindbrain.
Within the hindbrain, the fungal spores mimic local inflammation and attract immune cells, including macrophages, which phagocytose spores for their clearance.
While a few spores persist and germinate into hyphae, releasing pathogen-derived molecules and attracting neutrophils to the infection site.
Transfer an anesthetized larva into the medium-containing loading chamber of the zebrafish wounding and entrapment device for growth and imaging, or zWEDGI, device interconnected to the wounding chamber via a restriction channel.
Secure the larva within the restriction channel in a dorsal-lateral orientation for hindbrain imaging.
In the early stages, red fluorescence represents the fungal spores, while green fluorescence confirms macrophage recruitment to the infection site.
Over time, the spreading of red fluorescence signifies infection progression, while the emergence of additional blue fluorescence indicates neutrophil recruitment.
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.
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