Procedure
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.
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.