Double dye filling (using DiO and di-4 ANEPPS)

Double dye filling (using DiO and di-4 ANEPPS)
(Krista Williams)

Dye Filling

1. Label 15 ml centrifuge (c/f) tubes with strain name. Squirt ~1-2 ml M9 onto plate with a Pasteur pipet. Rinse plate with M9 using a pasteur pipet and transfer solution to c/f tube.
2. Spin pulse on high in a clinical c/f and carefully remove supernatant (s/n). Worm pellet will be very loose.
3. Wash once with M9 (optional).
4. Add 2 ml M9.
5. Add DiO (2 mg/ml in DMF) at 5 l/ml of M9.
6. Add di-4 ANEPPS (1mg/ml in DMF) at 8 l/ml of M9 and cover the c/f tube in foil to protect from light
7. Rock the tube for ~1 hour at 20°C on a lab quake rocker.
8. After rocking, spin pulse on high and remove s/n with Pasteur pipet.
9. Wash once with M9
10. Transfer worm pellet to labeled plate(s). Use enough plates that worms have sufficient food.
11. Allow the plates to sit at 20°C for ~20 minutes or sufficient time to pass the dye out of the digestive tract. NOTE: If worms are left too long, the di-4 ANEPPS will spread to all membranes in worm and make a very strong red background in whole body.

Viewing

1. Make pads using 5% agar with 10mM NaN₃ (10 l of 1M NaN₃ /ml of 5% agar -- our test tubes contain 4 ml of 5% agar).
2. Apply 4-6 l of M9 to one end of the pad.
3. Transfer 25-50 worms to the M9, adding more M9 if it starts to evaporate.
4. Put cover slip on and view with upright and fluorescence. Phasmid neurons should stain green (DiO) and phasmid socket cells should stain red (di-4 ANEPPS). Both structures should be visible through same filter. Limit exposure of worms to light, as di-4 ANEPPS is inactivated by light.