| Double dye filling (using DiO and di-4 ANEPPS)|
- 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.
- Spin pulse on high in a clinical c/f and carefully remove supernatant (s/n). Worm pellet will be very loose.
- Wash once with M9 (optional).
- Add 2 ml M9.
- Add DiO (2 mg/ml in DMF) at 5 l/ml of M9.
- 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
- Rock the tube for ~1 hour at 20°C on a lab quake rocker.
- After rocking, spin pulse on high and remove s/n with Pasteur pipet.
- Wash once with M9
- Transfer worm pellet to labeled plate(s). Use enough plates that worms have sufficient food.
- 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.
- 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).
- Apply 4-6 l of M9 to one end of the pad.
- Transfer 25-50 worms to the M9, adding more M9 if it starts to evaporate.
- 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.