We wish to thank S. Taneja-Bageshwar, K. Beifuss, S. Kedroske, and H-C. Hsiao for helpful discussions. This work was funded by start-up funds from the TAMHSC Department of Molecular and Cellular Medicine. The compound scope and spinning disk were purchased with funds provided by the department and the TAMHSC Office of the Dean. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Center for Research Resources. pRF4 (rol-6(su1006)) was a gift of A. Fire.

1. Preparation of DiI stain
<ol>
 <li>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.</li>
 <li>Create a working dilution of DiI by adding 0.6 &mu;L DiI stock to 399.4 &mu;L M9 for each population. This should give a final working dilution of 30 &mu;g/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.</li>
</ol>
<p class="jove_titl...

<ol>
	<li>Cox, G. N., Kusch, M., Edgar, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cuticle+of+Caenorhabditis+elegans:+its+isolation+and+partial+characterization.">Cuticle of Caenorhabditis elegans: its isolation and partial characterization.</a> The Journal of Cell Biology. 90, 7-17 (1981).</li><li>Cox, G. N., Staprans, S., Edgar, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cuticle+of+Caenorhabditis+elegans.+II.+Stage-specific+changes+in+ultrastructure+and+protein+composition+during+postembryonic+development.">The cuticle of Caenorhabditis elegans. II. Stage-specific changes in ultrastructure and protein composition during postembryonic development.</a> Dev. Biol. 86, 456-470 (1981).</li><li>Hall, D., Altun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans Atlas. , (2008).</li><li>Page, A. P., Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cuticle.">The cuticle.</a> The C. elegans Research Community. , (2007).</li><li>Kramer, J. M., Johnson, J. J., Edgar, R. S., Basch, C., Roberts, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sqt-1+gene+of+C.+elegans+encodes+a+collagen+critical+for+organismal+morphogenesis.">The sqt-1 gene of C. elegans encodes a collagen critical for organismal morphogenesis.</a> Cell. 55, 555-565 (1988).</li><li>Mende, N. v. o. n., Bird, D. M., Albert, P. S., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dpy-13:+a+nematode+collagen+gene+that+affects+body+shape.">dpy-13: a nematode collagen gene that affects body shape.</a> Cell. 55, 567-576 (1988).</li><li>Blaxter, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cuticle+surface+proteins+of+wild+type+and+mutant+Caenorhabditis+elegans.">Cuticle surface proteins of wild type and mutant Caenorhabditis elegans.</a> The Journal of Biological Chemistry. 268, 6600-6609 (1993).</li><li>Costa, M., Draper, B. W., Priess, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+actin+filaments+in+patterning+the+Caenorhabditis+elegans+cuticle.">The role of actin filaments in patterning the Caenorhabditis elegans cuticle.</a> Dev. Biol. 184, 373-384 (1997).</li><li>Sapio, M. R., Hilliard, M. A., Cermola, M., Favre, R., Bazzicalupo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zona+Pellucida+domain+containing+proteins,+CUT-1,+CUT-3+and+CUT-5,+play+essential+roles+in+the+development+of+the+larval+alae+in+Caenorhabditis+elegans.">The Zona Pellucida domain containing proteins, CUT-1, CUT-3 and CUT-5, play essential roles in the development of the larval alae in Caenorhabditis elegans.</a> Dev. Biol.. 282, 231-245 (2005).</li><li>Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cuticle+collagen+genes.+Expression+in+Caenorhabditis+elegans.">Cuticle collagen genes. Expression in Caenorhabditis elegans.</a> Trends Genet. 16, 21-27 (2000).</li><li>Kramer, J. M., French, R. P., Park, E. C., Johnson, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+rol-6+gene,+which+interacts+with+the+sqt-1+collagen+gene+to+determine+organismal+morphology,+encodes+a+collagen.">The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen.</a> Mol. Cell Biol. 10, 2081-2089 (1990).</li><li>Thein, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+exoskeleton+collagen+COL-19:+an+adult-specific+marker+for+collagen+modification+and+assembly,+and+the+analysis+of+organismal+morphology.">Caenorhabditis elegans exoskeleton collagen COL-19: an adult-specific marker for collagen modification and assembly, and the analysis of organismal morphology.</a> Dev. Dyn. 226, 523-539 (2003).</li><li>McMahon, L., Muriel, J. M., Roberts, B., Quinn, M., Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+sets+of+interacting+collagens+form+functionally+distinct+substructures+within+a+Caenorhabditis+elegans+extracellular+matrix.">Two sets of interacting collagens form functionally distinct substructures within a Caenorhabditis elegans extracellular matrix.</a> Molecular Biology of the Cell. 14, 1366-1378 (2003).</li><li>Link, C. D., Ehrenfels, C. W., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+expression+of+male+copulatory+bursa+surface+markers+in+Caenorhabditis+elegans.">Mutant expression of male copulatory bursa surface markers in Caenorhabditis elegans.</a> Development. 103, 485-495 (1988).</li><li>Tong, Y. G., Burglin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditions+for+dye-filling+of+sensory+neurons+in+Caenorhabditis+elegans.">Conditions for dye-filling of sensory neurons in Caenorhabditis elegans.</a> J. Neurosci. Methods. 188, 58-61 (2010).</li><li>Singh, R. N., Sulston, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+observations+on+moulting+in+Caenorhabditis+elegans.">Some observations on moulting in Caenorhabditis elegans.</a> Nematologica. 24, 63-71 (1978).</li><li>Collet, J., Spike, C. A., Lundquist, E. A., Shaw, J. E., Herman, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+osm-6,+a+gene+that+affects+sensory+cilium+structure+and+sensory+neuron+function+in+Caenorhabditis+elegans.">Analysis of osm-6, a gene that affects sensory cilium structure and sensory neuron function in Caenorhabditis elegans.</a> Genetics. 148, 187-200 (1998).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li></ol>

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 neurons15,17, 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...

<table border="1">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td>Synonyms</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>DiI</td>
			<td>1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate</td>
			<td>Biotium, Inc.</td>
			<td>60010</td>
			<td>Stock dilution: 
			20 mg/mL in DMF 
			working dilution: 30 mg/mL</td>
		</tr>
		<tr>
			<td>DMF</td>
			<td>Dimethylformamide</td>
			<td>Sigma-Aldrich, Inc.</td>
			<td>D4551</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton 
			X-100</td>
			<td>Octylphenoxypolyethoxyethanol</td>
			<td>VWR International, LLC.</td>
			<td>EM-9410</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>M9</td>
			<td>22mM KH2PO4, 42mM Na2HPO4, 86mM NaCl, 1mM MgSO4</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NGM</td>
			<td>Nematode growth medium</td>
			<td>IPM Scientific, Inc.</td>
			<td>11006-501</td>
			<td>Can be prepared following NGM agar protocol18</td>
		</tr>
		<tr>
			<td>Agar-agar</td>
			<td>&nbsp;</td>
			<td>EMD Chemicals, Inc.</td>
			<td>1.01614</td>
			<td>4% in water</td>
		</tr>
		<tr>
			<td>Levamisole</td>
			<td>Levamisole hydrochloride</td>
			<td>Sigma-Aldrich, Inc.</td>
			<td>31742</td>
			<td>100 &mu;M - 1 mM levamisole as required</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>&nbsp;</td>
			<td>VWR International, LLC.</td>
			<td>16005-106</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope cover glasses</td>
			<td>&nbsp;</td>
			<td>VWR International, LLC.</td>
			<td>16004-302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compound scope</td>
			<td>&nbsp;</td>
			<td>Carl Zeiss, Inc.</td>
			<td>A1m</td>
			<td>Use objectives to match the needs of the experiment</td>
		</tr>
		<tr>
			<td>TRITC or other compatible filter</td>
			<td>&nbsp;</td>
			<td>Chroma Technology Corp.</td>
			<td>49005</td>
			<td>ET - DSRed (TRITC/Cy3) sputtered filter set</td>
		</tr>
	</tbody>
</table>

visualization of caenorhabditis elegans cuticular structures using the lipophilic vital dye dii

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. 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 stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. 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.

Watch this Scientific Journal Video about Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI at JoVE.com

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

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.

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

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

visualization-caenorhabditis-elegans-cuticular-structures-using

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16.9K Views.  Texas A&M University System Health Science Center, College of Medicine. 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.

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

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...

combining lipophilic dye, in situ hybridization, immunohistochemistry, and histology

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

quantitative approaches for studying cellular structures and organelle morphology in caenorhabditis elegans

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

diagnosis of neoplasia in barrett&#8217;s esophagus using vital-dye enhanced fluorescence imaging

Diagnosis of Neoplasia in Barrett&#8217;s Esophagus using Vital-dye Enhanced Fluorescence Imaging

Vital-dye enhanced fluorescence imaging (VFI) is a novel in vivo technique that combines high-resolution epithelial imaging with exogenous topical fluorescent contrast to highlight glandular morphology and delineate neoplasia (high grade dysplasia and cancer) in the distal...

comprehensive assessment of germline chemical toxicity using the nematode caenorhabditis elegans

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid...

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

optimizing visualization of axonal transport of endogenous cargo by fluorescence microscopy in living caenorhabditis elegans

Author Spotlight: Advanced Techniques for Visualizing Endogenous Axonal Transport Dynamics

The paper describes the optimization of fluorescence microscopy acquisition parameters to visualize the axonal transport of endogenous labeled cargos at single-neuron resolution in a living...

Optimizing Visualization of Axonal Transport of Endogenous Cargo by Fluorescence Microscopy in Living Caenorhabditis elegans

identification of egfr and ras inhibitors using caenorhabditis elegans

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR...

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

dii perfusion as a method for vascular visualization in ambystoma mexicanum

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the...

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

super-resolution microscopy of the synaptonemal complex within the caenorhabditis elegans germline

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

Super-resolution microscopy can provide a detailed insight into the organization of components within the synaptonemal complex in meiosis. Here, we demonstrate a protocol to resolve individual proteins of the Caenorhabditis elegans synaptonemal complex.

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

assessing lysosomal alkalinization in the intestine of live caenorhabditis elegans

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

cultivation of caenorhabditis elegans in three dimensions in the laboratory

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

methodology to metabolically inactivate bacteria for caenorhabditis elegans research

Author Spotlight: Advancing Caenorhabditis elegans Research Using Paraformaldehyde-Treated Bacteria 

The food source for Caenorhabditis elegans in the lab is live Escherichia coli. Since bacteria are metabolically active, they present a confounding variable in metabolic and drug studies in C. elegans. A detailed protocol to metabolically inactivate bacteria using paraformaldehyde is described...

Bacterial Metabolic Inactivation in &lt;em&gt;Caenorhabditis elegans&lt;/em&gt; Using Paraformaldehyde

https://cloudfront.jove.com/CDNSource/teasers/65775_b.jpg

isolation and characterization of the natural microbiota of the model nematode caenorhabditis elegans

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

using caenorhabditis elegans to screen for tissue-specific chaperone interactions

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

methods for studying the mechanisms of action of antipsychotic drugs in caenorhabditis elegans

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than...

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

characterization of amyloid structures in aging c. elegans using fluorescence lifetime imaging

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease...

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

imaging dendritic spines in caenorhabditis elegans

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or...

Imaging Dendritic Spines in Caenorhabditis elegans

examining the effect of pesticides on caenorhabditis elegans neurons

Examining the Effect of Pesticides on Caenorhabditis elegans Neurons

Young adult Caenorhabditis elegans nematodes are exposed to different concentrations of commercial pesticides or other toxicants for 2-24 h. Then, different neurons can be visualized using fluorescent-expressing strains. This paper demonstrates how to expose nematodes to pesticides and assess neuron damage.

Examining the Effect of Pesticides on Caenorhabditis elegans Neurons

measuring the effects of bacteria and chemicals on the intestinal permeability of caenorhabditis elegans

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel...

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

analysis of transgenerational epigenetic inheritance in c. elegans using a fluorescent reporter and chromatin immunoprecipitation (chip)

Author Spotlight: RNAi Inheritance and ChIP in C. elegans 

This protocol describes an RNA interference and ChIP assay to study the epigenetic inheritance of RNAi-induced silencing and associated chromatin modifications in C. elegans.

Epigenetic Inheritance and ChIP in &lt;em&gt;Caenorhabditis elegans&lt;/em&gt;

https://cloudfront.jove.com/CDNSource/teasers/65285_b.jpg

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Hand Dissection of Caenorhabditis elegans Intestines

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, aging, immunity, and environmental response. Critical interactions between the C. elegans host and its environment converge within the intestine, where gut microbiota concentrate. Therefore, the ability to isolate intestine tissue away from the rest of the worm is necessary to assess intestine-specific processes. This protocol describes a method for hand dissecting adult C. elegans intestines. The procedure can be performed in fluorescently labeled strains for ease or training purposes. Once the technique is perfected, intestines can be collected from unlabeled worms of any genotype. This microdissection approach allows for the simultaneous capture of host intestinal tissue and gut microbiota, a benefit to many microbiome studies. As such, downstream applications for the intestinal preparations generated by this protocol can include but are not limited to RNA isolation from intestinal cells and DNA isolation from captured microbiota. Overall, hand dissection of C. elegans intestines affords a simple and robust method to investigate critical aspects of intestine biology.

Hand Dissection of Caenorhabditis elegans Intestines

The present protocol describes a procedure for isolating intestines from adult Caenorhabditis elegans nematode worms by hand for input in genomics, proteomics, microbiome, or other assays.

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, ...

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures the interaction of homologous chromosomes is the assembly of the synaptonemal complex (SC) in meiotic prophase I. Even though there is little sequence homology between protein components within the SC among different species, the general structure of the SC has been highly conserved during evolution. In electron micrographs, the SC appears as a tripartite, ladder-like structure composed of lateral elements or axes, transverse filaments, and a central element. 
However, precisely identifying the localization of individual components within the complex by electron microscopy to determine the molecular structure of the SC remains challenging. By contrast, fluorescence microscopy allows for the identification of individual protein components within the complex. However, since the SC is only ~100 nm wide, its substructure cannot be resolved by diffraction-limited conventional fluorescence microscopy. Thus, determining the molecular architecture of the SC requires super-resolution light microscopy techniques such as structured illumination microscopy (SIM), stimulated-emission depletion (STED) microscopy, or single-molecule localization microscopy (SMLM). 
To maintain the structure and interactions of individual components within the SC, it is important to observe the complex in an environment that is close to its native environment in the germ cells. Therefore, we demonstrate an immunohistochemistry and imaging protocol that enables the study of the substructure of the SC in intact, extruded Caenorhabditis elegans germline tissue with SMLM and STED microscopy. Directly fixing the tissue to the coverslip reduces the movement of the samples during imaging and minimizes aberrations in the sample to achieve the high resolution necessary to visualize the substructure of the SC in its biological context.

Super-resolution microscopy can provide a detailed insight into the organization of components within the synaptonemal complex in meiosis. Here, we demonstrate a protocol to resolve individual proteins of the Caenorhabditis elegans synaptonemal complex.

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures ...