To compare antibody staining over time, one needs to have a method of archiving tissue samples. In general, however, C. elegans prepared for histology have not been archived for long periods. For example, worms mounted on slides sealed with nail polish and stored at 4°C (Wilson, et al., 2012; Duerr, 2013) last two weeks to a month (Duerr, 2013). After this time, significant tissue shrinkage occurs and fluorescent signals rapidly decay (Figure 1A). I have found that the polyvinyl pyrrolidone-based cryoprotectant known as “anti-freeze” (Tebbenkamp, et al., 2011) that is used to archive partially and fully processed mammalian tissues for long periods works well with histologically prepared C. elegans.

I prepared animals using the staining method of Finney and Ruvkun (Finney and Ruvkun, 1990) with antibodies against MEC-18 and MEC-2 or the 4′,6-diamidino-2-phenylindole (DAPI) probe against DNA or the actin-binding reagent phalloidin. Animals were then archived with or without anti-freeze for varying times. In contrast to sealing with nail polish no significant degradation of tissue morphology or fluorescence was seen for at least a year (Figure 1B-C). Similar results were observed with worm cell cultures. Thus, anti-freeze did not disrupt antibodies bound to their epitopes. In addition, animals expressing GFP and mCherry that were observed after several months in anti-freeze remained intensely fluorescent as were animals stained following long-term storage in anti-freeze. In general, the morphology of touch receptor neurons and other cells was indistinguishable in worms that were archived in anti-freeze compared to worms that were immediately analyzed. Moreover, morphology remained intact even after several freeze/thaw cycles.

To archive, mix 50 ml worm pellet and 500 ml anti-freeze (100 mM sodium acetate, 250 mM polyvinyl pyrrolidone (PVP), and 40% ethylene glycol, pH 6.5), and store at -20°C. The anti-freeze itself can be stored for years at 4°C or -20°C.

To recover worms for histology, pellet animals and wash 2-3 X with M9 buffer.

Drugs are chemical substances of natural or synthetic origin used for diagnosis, prevention, treatment, and control of disorders. Most drugs are specific to control and cure diseases and, although their curing effects outweigh their side effects, most are associated with side effects. One example is aspirin, which is a salicylate drug, prescribed as an analgesic, antipyretic, and anti-inflammatory medication; it has been reported to cause Reye syndrome (sudden brain damage and liver problems) when given to children suffering from flu and chicken pox (Belay et al., 1997). The other side effects of aspirin are gastrointestinal ulcers and stomach bleeding (Sorensen et al., 2000).

C. elegans has a well conserved IGF-1/insulin-like signaling pathways. daf-2 encodes an insulin-like receptor in C. elegans, which is a orthologue of the mammalian insulin/IGF receptor family (Kimura et al. 1997). In daf-2(e1370) animals, the decrease of insulin-like signaling results in increased dauer formation. Quantification of daf-2(e1370) dauer formation in C. elegans can be used as an index to measure the level of IGF-1/insulin signaling: enhancement of IGF-1 signaling causes increased dauer formation while decrease in IGF-1 signaling causes cessation in number of dauers formed.

We examined the effect of the drugs silymarin, Godex, aspirin, and pravastatin in modulating IGF-1/insulin signaling. Effects of these drugs on IGF-1/insulin signaling have not been explored. Silymarin is a lipophilic flavonolignan (flavanone derivative) extracted from the seeds and fruits of Silybum marianum (milk thistle). Godex is composed of carnitine orotate, which is the major chemical constituent, it also contains seventeen amino acids as well as vitamin B2, B6, and B12. Both silymarin and Godex are known hepatoprotectant drugs and are mainly used in the clinical treatment of liver cirrhosis, acute liver intoxication, and chronic hepatitis (Al Anati et al., 2009; Park et al., 2001). Aspirin is a salicylate drug used as an analgesic, antipyretic, and anti-inflammatory medication. Pravastatin, a statin, is issued for lowering cholesterol and preventing cardiovascular disease, and is generally well tolerated (Pfeffer et al., 2002).

To test the effect of the above mentioned drugs on IGF-1/insulin signaling, we used daf-2(e1370) animals. Synchronized eggs of daf-2(e1370) were seeded on drug treated (100 µM) and control plates at 22ºC. After 4 days of incubation the number of dauers present on the respective plates were counted manually and further confirmation of dauer was done by SDS treatment. The mean percentage ± S.D of dauer formation on control plates was 41.8±6.1 whereas the drug treated plates showed respective percentage of 1.8±0.7 (p<0.0001) for silymarin, 4.9±1.2 (p<0.0001) for aspirin, and 23.6±2. (p<0.0001) for Godex. All the three drugs showed a statistically significant effect on suppressing dauer formation. The mean percentage of dauer formation on pravastatin treated plate was 78.8 ±11.9, which was also statistically significant (p<0.0001) as compared to control (41.8±6.1).

Our results show that both the hepatoprotective drugs silymarin and Godex, as well as the well-known analgesic drug aspirin, suppress the IGF-1/insulin signaling pathway, while the cholesterol-lowering drug pravastatin enhances this pathway. Our results thus suggest that daf-2(e1370) animals can be used to examine the effect of pharmacological drugs on IGF-1/insulin signaling. This strategy might be useful to examine the effect of drugs in relation to IGF-1 signaling and can be further extrapolated to study the effect of different drugs in relation to insulin signaling in human subjects.

Cell specific reporters are great tools to visualize cell and developmental processes in living animals as their characteristics can be easily identified, selected, or measured. For generation of mutant strains containing integrated GFP reporters, it is required to determine whether or not the desired strain is obtainable by setting up the necessary crosses and following the progeny. If the reporter and the mutation are located closely, it would be difficult to combine them and isolate the desired genotype. Lateral hypodermal seam cells are epithelial stem cells in C. elegans which have been utilized as a great tool to study numerous biological processes such as cell fate, cell signaling, asymmetric division, cell polarity and developmental timing. Seam cell specific transcriptional GFP reporter (scm_p::GFP, wIs51) which expresses GFP in all seam cells at all developmental stages, was originally made by M. Fukuyama and Jodie J. Kasmir in Joel Rothman laboratory (Terns et al., 1997; Koh and Rothman, 2001) and has been utilized in numerous studies. However, although the insertion site of wIs51 is previously reported to be on chromosome V, its precise location is not known. Here we report that wIs51 is closely linked to unc-76.

During a series of genetic crosses between double mutant unc-76(e911) lin-46(ma164) and wIs51 we obtained the recombinant animals unc-76(e911) lin-46(ma164) wIs51 at 1/46 (0.022) frequency. Therefore, the genetic distance between unc-76(e911) 46(ma164) and wIs51 is 2% or 2 centiMorgan on chromosome V. Worms are diploid and progeny will have a chance to receive such a recombinant chromosome from either oocyte or sperm. Thus, the chance of progeny receiving two recombinant chromosomes will be quite small. Further evaluations confirmed the presence of lin-46(ma164) by DNA sequencing in addition to the presence of GFP (wIs51) and uncoordinated phenotype of unc-76 in the recombinant animals. More precise mapping, such as physical mapping would be necessary to identify the exact physical location of wIs51. This finding will be informative to those who are planning to incorporate wIs51 to their C. elegans strains.