Fat bodies are dissected in Schneider’s drosophila medium (1×);

Fixed in 4% paraformaldehyde (PFA) for 45 min at room temperature (ask Mark).

Samples are rinsed, washed with 0.1% Triton X-100 in 1× PBS (1× PBT) (3 times, 10 min/time).

Blocked in 5% horse serum in 1× PBT at 4°C overnight.

Incubate primary antibodies (Rat-anti-TIM, 1:500; Guinea pig-anti-PER, 1:500) at 4°C overnight.

Washed with 1× PBT for 1 hour.

Secondary antibodies (Rat-Alexa 488, 1:500; Guinea pig-Alexa 555, 1:500) were incubated at 4°C overnight.

Washed with 1× PBT for 1 hour.

Stain for 15 min with DAPI (Sigma; 0.1 μg/ml)

Remove DAPI and add PBT.

The samples were mounted in mounting medium mowiol (ask Mark).

All images were captured by a Zeiss LSM780 inverted confocal microscope and were processed in Adobe Photoshop and Illustrator.