DiI and DiO Staining in C. elegans

1. DiI Staining of Amphid (ADL, ASH, ASI, ASJ, ASK, AWB), Phasmid (PHA and PHB), IL1 and IL2 neurons and IL Sheath and Socket cells

1. A stock dye solution containing 2mg/ml DiI (Molecular Probes, catalog # D-282) in dimethyl formamide can be stored at -20°C in a tube wrapped in foil.
2. Transfer well-fed worms from a plate into an eppendorf tube with 1 ml M9, spin worms down at 2000-3000 rev/min, take out supernatant leaving (loose) worm pellet.
3. Resuspend worms in1 ml M9 and add 5 microliter DiI stock sol (1:200 dilution) and incubate on a slow shaker for 3 hr-overnight (some dye may precipitate).
4. Spin and wash worms with M9 twice before transferring them onto agar pads with sodium azide to visualize by fluorescence using the appropriate filters (DiI fluoresces red, therefore use the Texas red filters on the fluorescence scope).
5. To additionally stain inner labial neurons and inner labial socket and sheath cells with DiI, wash the worm plate with 1ml H2O/50 mM Calcium Acetate, wash once with 50 mM Calcium acetate and resuspend worms in 1ml H2O/50 mM Calcium Acetate.
6. Add DiI to this solution and incubate overnight. At the end of staining, wash with H2O before transferring worms onto agar pads with azide.

Pictures of DiI Staining Patterns:

Amphids in D-V view
Amphids in lat view
Phasmids in D-V view
Inner labial neurons and inner labial socket and sheath cell

2. Protocol for DiI and DiO Staining of Amphid (ADL, ASH, ASI, ASJ, ASK, AWB) and Phasmid (PHA and PHB) Neurons by Michael Koelle

1. Buy DiO from Molecular Probes, catalog # D-275. DiO fluorescesces green (use FITC filters). If you want red fluorescence, DiI can be substituted; stain exactly the same way but use the Texas red filters on the fluorescence scope. DiI may stain a slightly different set of cells.
2. Stock solution is 2 mg/ml in dimethly formamide, stored at -20°C in a foil wrapped tube.
3. Dilute the stock 1:200 in M9. Some dye will precipitate when you do this; don't worry about it.
4. Put 150 l stain in a microtiter well, and use a worm pick to put transfer some worms into the dye. Incubate 2-3 hours at room temperature.
5. Use a mouth pipette to transfer the worms to a fresh plate, and let them crawl on a bacterial lawn for about 1 hour to destain. (Alternatively, wash the worms 3X in M9.)
6. Put worms on agar pads with sodium azide and visualize by fluorescence using the appropriate filters.

3. Dye-filling with DiO by Ed Hedgecock (modified by Herman, Williams and Hettenbach)

4. Modified DiI Staining Procedure to Stain IL2 Neurons by Tong and Bürglin:

Tong, Y-G., and Bürglin, T.R. 2010. Conditions for dye-filling of sensory neurons in Caenorhabditis elegans. J. Neurosci. Method. 188: 58-61. Article
(this protocol exaimines the conditions under which the IL2 neurons best take up the lipophilic fluorescent dye)

5. Staining Cuticular Structures with DiI by Schultz and Gumienny

Schultz, R.D. and Gumienny, T.L. 2012. Visualization of Caenorhabditis elegans cuticular structures using the lipophilic vital dye DiI. J. Vis. Exp. 59: e3362. Article with video
(this optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans).

6. See also Cell Biology and Methods in Cell Biology Chapters in Wormbook