Procedure
Stock Solution:
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
Method:
Dissect flies in PBS
Fix with 250-300 ul of 4% PFA in PBS for 30 min at room temperature (RT)
100 mg PFA + 1 ul NaOH + 500 ul H2O --> 20% stock (2-3 min @ 65°C to dissolve)
50 ul of 20% stock to 200 ul PBS
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]
align all tissues facing up
1x drop of medium (~35ul]
add cover slip