Protocol for Nile Red Staining - Fly

PBS

Fix solution (freshly prepared): 4% paraformaldehyde (PFA) in PBS

PBX: PBS, 0.1% Triton X-100

Nile Red (500 ul/ml) dissolved in acetone 

Dissect flies in PBS 

Fix with 250-300 ul of 4% PFA in PBS for 30 min at room temperature (RT) 

Wash with 300 ul of PBS for 10 min at RT x3 times. 

Incubate with 250 ul of 1 ug/ml Nile Red (0.5 ul NR/sample, 250 ul) in PBX for 2 hrs at RT or overnight at 4°C 

Wash with 300 ul of PBX for 5 min at RT x2 times 

Incubate with 300 ul of DAPI in PBX (1:4,000 dilution) for 20 min at RT 

Wash with 300 ul of PBX for 10 min at RT x3 times 

Wash with 300 ul of PBS for 5 min at RT x2 times 

Mount on the slide glass with Mowiol medium [M]