This article describes a method for imaging genetically modified Purkinje neurons in transgenic zebrafish larvae. The technique involves anesthetizing the larvae, immobilizing them in agarose, and using confocal microscopy to visualize neuronal activity.
Begin with anesthetized transgenic zebrafish larvae in a mounting chamber containing an oxygenated physiological buffer under a stereomicroscope.
The cerebellum in the brain contains genetically modified Purkinje neurons expressing fluorescent cytoplasmic proteins.
Remove excess medium, then add molten agarose to immobilize the larvae for stable imaging.
Orient the larvae dorsal side up to visualize the cerebellum.
Once solidified, trim excess agarose. Using a glass needle, excise the pigmented skin overlying the brain to expose it.
Flip the agarose block onto an imaging chamber containing molten agarose for imaging under an inverted microscope.
Add the physiological buffer to preserve neuronal function.
Position the chamber under an inverted confocal microscope. Apply excitation light to excite the fluorescent proteins, enabling visualization of the labeled neurons.
The pigmented skin cells, if not removed, scatter and absorb the excitation light, reducing fluorescence intensity.
Skin removal improves light penetration to enhance fluorescence intensity, facilitating neuron visualization.
To embed the larva, use a Pasteur pipette to transfer the anesthetized larva to a mounting chamber under a stereomicroscope. If any larva are still able to move, do not use the fish for the experiment until the fish are completely unable to move. When the larva are immobile, carefully remove the excess medium and immediately add at least 1 milliliter of low-melting agarose onto the larva.
Orient the zebrafish with the dorsal region facing up as close to the surface of the agarose as possible. If the larvae will be imaged using an inverted microscope, after solidifying, trim the agarose containing the larva into a small cuboid block. To expose the brain for imaging, trim away the excess agarose over the brain region of interest as necessary, and use a glass needle to make a small incision through the skin near but not over the region of interest, without penetrating too deeply into the tissue.
Barely moving the needle just under the skin surface, continue to carefully make very small cuts around the region of interest until the skin over the region of interest can be removed or pushed aside. When the tissue has been removed from all of the embryos, add a small drop of low-melting agarose to a previously prepared imaging chamber of an inverted microscope, and use a small spatula to flip the cuboid agarose block 180 degrees onto the agarose drop in the imaging chamber. When the agarose has solidified, fill the imaging chamber with fresh ACSF and begin imaging the larva.
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