To begin, mix the blue soft gel paste food coloring with distilled water. Microwave the food vials containing standard cornmeal agar food until the food has melted into a liquid. Add the dye mixture to each vial of food and mix well until the food is homogeneously blue.
Add an amount of blue dye mixture to saturate the color of the food, ensuring no variation in color between the vials. Place a thin paper towel over vial openings during drying to prevent any stray flies from landing in the vial. Once the food is cooled and solidified, transfer the flies to the blue food containing vial.
Incubate the vials with flies overnight at 25 degrees Celsius. The next day, transfer the flies to vials containing standard Drosophila food and number each vial. Incubate the flies at 25 degrees Celsius for 60 minutes.
After incubation, transfer the flies to new vials with standard Drosophila food. Draw a dotted line down the length of the vial to mark the starting point for counting. Manually count the small, round, opaque and colorless dots on the wall of each vial.
Record the ratio of blue dots to the total number of dots for each vial. In control flies, the blue food is expelled relatively quickly while in alpha-synuclein flies, the blue food remains in the gut for up to eight hours. At day 10 post-eclosion, when the alpha-synuclein flies display a robust neurodegenerative phenotype, significant differences in gut transit time compared to control flies were observed.
However, no differences in gut transit were observed between control and alpha-synuclein flies on day one post-eclosion.
