Visualizing Mushroom Body Neurons in Drosophila Brains via Immunostaining

Place wild-type and mutant Drosophila brains in a solution containing a fixative and a detergent. Incubate with gentle rocking.

The fixative preserves the tissue morphology, while the detergent permeabilizes cell membranes.

Allow the brains to settle, then remove the solution.

Wash the tissues with a buffer.

Add a blocking solution to prevent non-specific antibody binding.

Remove the solution and add primary antibodies targeting a specific protein that regulates axonal development in the brain's mushroom body, or MB, neurons.

Incubate to promote antibody binding.

Wash with buffer to remove unbound antibodies.

Incubate with fluorophore-conjugated secondary antibodies targeting the primary antibodies.

Wash again to remove unbound antibodies, then resuspend the brains in a mounting medium.

Mount the brains on a bridge slide and visualize them under a fluorescence microscope.

Compared to the wild-type, the mutant brains exhibit a defective phenotype, with axonal mis-projections and missing MB lobes.

Under the microscope, use a P200 pipette to transfer 10 to 15 dissected brains at the same genotype into a 0.5-milliliter microcentrifuge tube filled with 4% paraformaldehyde diluted in PTN