We are having some trouble with our secondary antibodies for IF giving high background, especially Cy3 conjugated versions. We are considering using an acetone powder to clean up our secondaries vs. nematode antigens. If anyone has any other suggestions, or secondaries that have low background on worm embryos, I’d appreciate it. steve
Steve,
You can fix a bunch of N2 with the same procedure that you use to fix your worms for staining. Then, you can incubate the secondary antibodies with the fixed worms. This will remove any secondary antibodies that bind to the worms non-specifically. See the WormBook chapter on Immunohistochemistry (protocol 2.12) for details.
If you would prefer to try a different secondary, I have had good luck with Cy3-Donkey secondary antibodies from Jackson ImmunoResearch Labs. I use the more expensive, but cleaner “ML” ones that have had cross-reactivity to other species removed (e.g., .Donkey Anti-Rabbit IgG (H+L) ML #711-165-152).
Good luck,
Janet Duerr