Imaging and Quantification of Protein Aggregates in Genetically Modified Drosophila Brains

Begin with genetically modified Drosophila brains. These brains express a red fluorescent-tagged mutant protein in the neuron that can spread to the surrounding glial cell, expressing the yellow fluorescent-tagged normal protein, causing their aggregation.

Add a fixative solution that preserves cellular integrity.

Remove the fixative and wash with a buffer.

Add an antifade reagent and incubate to prevent the fading of fluorescent signals.

Transfer the brains onto a slide and remove excess liquid.

Allow the brains to adhere to the slide.

Using small pieces of a coverslip as spacers, place a coverslip on top of them. Add antifade reagent and seal it.

Under a confocal microscope, capture images using different excitation wavelengths for red and yellow fluorescence.

Using image analysis software, quantify the protein aggregates.

Co-localization of red and yellow fluorescence indicates the spreading of mutant proteins.

Once all of the brains have been dissected, transfer the collection tube to a nutator at room temperature and rock them for about 5 minutes. After the fixation period, remove the majority of the fixative solution using a P1000 pipette and discard it, being very careful to