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 leave the brains in the tubes. This requires gentle suction and careful observation.

Next, add 1 milliliter of fresh PBST to the brains. Cover up the tube and let it rock on the nutator for about a minute to wash off the remaining fixative. Then, remove the solution and repeat this short wash step.

Follow the two short washes with one 5-minute wash, then three 20-minute washes, and, finally, a single 1-hour wash. After the last wash, carefully remove most of the PBST and submerge the brains in 30 microliters of glycerol-based anti-fade reagent. Then, incubate the brains at 4 degrees Celsius in the dark without movement for 1 to 24 hours.

Later, remove the brains from the collection tube using a blunted pipette tip and transfer them to a microscope slide. Then, gently orient the brains as needed for imaging using forceps. Multiple samples can be mounted on the same slide in separate rows.

Next, remove the excess anti-fade reagent from the slide using the corner of a folded lab tissue. Do not let the tissue come into contact with the brains. Then, leave the samples in the dark for 5 to 10 minutes to let the brains adhere to the slide.

Next, take small pieces of broken cover glass and position them around the brains, covering an area that is about 19 square millimeters. Then, gently lower a 22-square-millimeter cover glass over the mosaic of brains and glass to make a bridge mount. Next, slowly dispense fresh anti-fade reagent under the coverslip so it fills in the empty spaces. Do this very carefully so that the brains and glass stay in place. Then, seal the coverslip with nail polish. First, just apply it at the corners. Let the corner dabs dry 5 to 10 minutes before completing the seal along the edges. The brains should be imaged as soon as possible.

Image the mounted brains using a confocal microscope equipped with a 40x or 63x oil objective to collect z-slices in the region of the brain where the transgenes are expressed. Then, analyze the data by quantifying the puncta in the individual z-slices or, alternatively, after rendering the slices in three dimensions.

To quantify mutant Huntington aggregates which are well-separated and have little background signal, open the confocal z-series in the 3D Viewing Mode. Then, use the Analysis Wizard to identify individual spots in a selected channel.

In the settings, adjust the thresholding and filters to accurately represent all heterogeneously sized aggregates as individual objects in the image. Then, enable Split Objects under Binary Processing Pre-Filter to separate closely associated aggregates. Quantitative information about the objects identified by the software is reported under Measurements.

After counting the puncta, further characterize them in image analysis software. For example, take relevant measurements of the spots or surfaces to obtain aggregate diameter, volume, or intensity information. Some wild-type Huntington aggregates can be quantified by manually moving through the z-stack and counting green puncta that are distinguishable from the surrounding diffuse signal.

Be careful to avoid counting single aggregates twice when they appear in more than one slice. Another analysis of interest is determining the frequency of co-localization between Huntington Q25-YFP and Huntington Q91-mCherry aggregates. Do this using manual counting by moving slice by slice through a confocal z-stack.