In Vivo Imaging of Zebrafish Larval Brain for Visualizing Cerebellar Neurons

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 unde