Ecdysone was purchased in powder form and was dissolved in ethanol. Then it was mixed with 2% agarose containing 5% sucrose to make the different doses of ecdysone tubes, and the same amount of ethanol as in the ecdysone solution was used to make the vehicle control tubes. Only 2% agarose and ecdysone were used to make ecdysone starvation tubes. Five- to seven-day-old flies were loaded into locomotor tubes for ecdysone treatment and behavior recording to verify that ecdysone promotes sleep in ecdysone-treated flies. For ecdysone feeding/starvation assay, all flies were kept in normal tube on days 0, 1, and 2. Flies were then transferred to either starvation tubes, or ecdysone starvation tubes between ZT23 and ZT0 on day 2. Flies were kept in these ecdysone/starvation tubes for 1 day and transferred back to normal tubes again between ZT23 and ZT0 on day 3 and recorded for another day. Data from days 2 to 4 were used for sleep analysis. For gaboxadol hydrochloride treatment, it was dissolved in water and diluted to 0.1 mg/ml as final concentration for locomotor tube experiments. Sleep was measurement from days 2 to 4, after flies were loaded into gaboxadol tubes at day 0. For TARGET system experiments, flies were raised at permissive temperature 18°C, and 5- to 7-day-old adult flies were used to loaded and were kept at 18°C from days 0 to 2, 31°C from days 3 to 4, and 18°C for day 5. Data from days 2 to 5 were collected for sleep analysis.
BODIPY493 was used for brain LD staining. Flies were loaded into locomotor tubes and subjected to 0.5 mM ecdysone treatment for 12 or 36 hr. Both control and ecdysone-treated flies were then dissected in the phosphate-buffered saline (PBS), fixed in 4% Paraformaldehyde (PFA) solution for 20 min, and washed three times with PBS + 0.3% Triton (PBT). Then brains were left in PBT at 4 degrees overnight and transferred to 1 µg/ml BODIPY493 in PBT for 20 min. Later they were mounted for imaging.
Brains were imaged with the oil-immersion ×40 lens of a confocal microscope at a resolution of 1024 × 1024. Raw images were processed with FIJI ImageJ. The first step was to remove non-lipid trash and exclude artificial signals or signals outside the brain area. The second step was to quantify the LD count, area, and total brain tissue area. Later, the LDs count will be normalized by the brain tissue area, and LD size calculated by LD area divided by total count. Quantifications were conducted by using ImageJ Macro.
BODIPY493 was used for brain LD staining. Flies were loaded into locomotor tubes and subjected to 0.5 mM ecdysone treatment for 12 or 36 hr. Both control and ecdysone-treated flies were then dissected in the phosphate-buffered saline (PBS), fixed in 4% Paraformaldehyde (PFA) solution for 20 min, and washed three times with PBS + 0.3% Triton (PBT). Then brains were left in PBT at 4 degrees overnight and transferred to 1 µg/ml BODIPY493 in PBT for 20 min. Later they were mounted for imaging.
Brains were imaged with the oil-immersion ×40 lens of a confocal microscope at a resolution of 1024 × 1024. Raw images were processed with FIJI ImageJ. The first step was to remove non-lipid trash and exclude artificial signals or signals outside the brain area. The second step was to quantify the LD count, area, and total brain tissue area. Later, the LDs count will be normalized by the brain tissue area, and LD size calculated by LD area divided by total count. Quantifications were conducted by using ImageJ Macro.
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