Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Summary

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

Abstract

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal^1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility^4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer⁷.

Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development⁸. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages^2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology^5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression¹², antibody staining¹³, and electron microscopy¹. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures¹⁴. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail¹⁵. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. 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.

Protocol

1. Preparation of DiI stain

1. Prepare a stock solution of 20 mg/mL DiI (Biotium, Inc., Hayward, CA) in DMF. DiI is light sensitive, so protect DiI from light by wrapping in foil.
2. Create a working dilution of DiI by adding 0.6 μL DiI stock to 399.4 μL M9 for each population. This should give a final working dilution of 30 μg/mL DiI in M9. This can be scaled up for staining multiple populations simultaneously. Shield DiI from light by wrapping the tube(s) in foil.

2. Preparation of nematodes

1. Use a 60 mm plate containing a population of uncontaminated nematodes. Wash animals from plate using a solution of....

Discussion

The DiI staining method presented here allows for a relatively quick and convenient way to visualize the cuticle in C. elegans. By repurposing and optimizing a method commonly used to image environmentally exposed sensory neurons^15,17, DiI can be used to fluorescently stain both alae and annular structures (Figures 1 and 2), as well as the vulva, male tail, and hermaphrodite tail spike (Figure 3). We have found that the incubation solution and time influence the ability of DiI to consistently stain th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DiI (1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate)	Biotium, Inc.	60010	Stock dilution: 20 mg/mL in DMF working dilution: 30 mg/mL
DMF (Dimethylformamide)	Sigma-Aldrich	D4551
Triton X-100 (Octylphenoxypolyethoxyethanol)	VWR international	EM-9410
M9 (22mM KH2PO4, 42mM Na2HPO4, 86mM NaCl, 1mM MgSO4)
NGM (Nematode growth medium)	IPM Scientific, Inc.	11006-501	Can be prepared following NGM agar protocol18
Agar-agar	EMD Millipore	1.01614	4% in water
Levamisole (Levamisole hydrochloride)	Sigma-Aldrich	31742	100 μM - 1 mM levamisole as required
Microscope slides	VWR international	16005-106
Microscope cover glasses	VWR international	16004-302
Compound scope	Carl Zeiss, Inc.	A1m	Use objectives to match the needs of the experiment
TRITC or other compatible filter	Chroma Technology Corp.	49005	ET - DSRed (TRITC/Cy3) sputtered filter set