Hi All
Hope you can advise.
I’m trying to do some immunohistochemistry on a couple stages of a parasitic nematode: adults and infective larvae.
We have a protocol for chemically permeabolizing the adults (based upon the C. elegans protocol that uses BetaMercaptoEthanol and Collagenase treatment) but we do not know if this will work with our infective larvae, which has a much thicker cuticle (similar to dauer stage of C. elegans). Hence, I’ve been trying to find some C. elegans protocols for immunohistochemistry of the dauer stage with no success. WormBook lists a few fixation protocols, but do not specify whether they work well on the dauer stage. Have any of you tried fixing dauers? Or come across some protocols which would be a nice starting point?
I realize that your question is only in regards to LM immunochemistry. However, I will give a report for EM methods anyway. The basic lesson is that you are correct to be worried about the extra cuticle, but you should be able to adapt a previous protocol to get a positive result.
We have done EM immunocytochemistry on several other nematode species, such as hookworms, which have a dauer-like cuticle (unpublished work). We use the same microwave fixation and post-embedding steps as we published in Paupard et al., 2001 for C. elegans with one major exception.
Due to the thicker cuticle, we needed to double all the microwave steps to coax fixatives to cross into the animal. Repeat the microwave processes over again at each step, using the same techniques to limit specimen heating. If you have access to the old style Pella microwave oven, you can follow those steps exactly, placing the specimens in a shallow well, cooled by ice slush at a “hot spot” in the oven. This microwave irradiation also facilitates the dehydration steps and resin infiltration across the dauer cuticle. Otherwise dauers will look crushed or empty due to lack of fluid exchange.
If you have access to the newer Pella “Biowave” oven, there are no hot spots. Instead we now use their “Coldspot” device, giving uniform microwave energy across the whole chamber. For those purposes, the same basic steps should still work, but you may need to extend the exposure times still longer. For that purpose, we now adjust the power setting of the Biowave oven very low (only 70 watts), which means you can irradiate the sample for 15 min or more without hitting the restriction temperature (which we set at 39oC).
I will try to give updated protocols soon on the WormAtlas Methods section. We presented some of these results recently at the MSA meeting in Florida.