I am trying to use Acridine Orange to stain apoptotic oocytes in the germline of N2 hermaphrodites using this protocol: Fluorescent Visualization of Germline Apoptosis in Living Caenorhabditis elegans, by Benjamin Lant and W. Brent Derry. It worked once but I have not been able to get it to work since then.
We have tried decreasing the clearing time and screening for corpses before mounting, with no success. The stain works and is visible in the gut, but not the germline.
My next idea was to increase the feeding time on the stain from 1 hour to 2 hours, and increase the number of worms picked to increase odds of success. Any other suggestions?
The one successful stain was also a failure in terms of data because the AO photobleached so quickly I could not get a quality photo. Is that a common issue or is there a way to increase the life of the stain?
Thank you.
Hi,
AO is good and I have seen it work well. However, I would suggest looking into the CED-1::GFP expressing strain MD701. It is a good marker for apoptosis. See one of our papers using it here: " Cap-Independent Translation Promotes C. elegans Germ Cell Apoptosis through Apaf-1/CED-4 in a Caspase-Dependent Mechanism"
Cheers,
Hayden Huggins