So I have been having serious problems getting a monoclonal engrailed antibody to work in a megaselia species. The antibody was designed in drosophila, but is cross reactive. It just takes (and this seems to be about the same in droso as well, the dillution not the rest) a 1:5 primary dillution for two days at 4 degrees. Using just a standard flourescent labeled secondary at 4hrs after ward gets a really weak signal. trying to image, the exposure was about 1.5 seconds, which is sort of absurd. I've tried TSA and it didn't work. I tried colorimetric using an HRP conjugated secondary with a 10 minute rx time and an hour rx time and neither worked. Neither of these methods produced ANY signal.
I'm just trying to figure out some way to make this work. But, beyond maybe the antibody just doesn't penetrate well, I can't think of anything that would cause this sort of complete failure. Especially since the regular secondary sort of worked, it's amazing to me that the TSA and the colorimetric didn't produce anything. Even sticking with the regular secondary, I don't know if incubating it for longer, say 2 days primary and 1 day secondary both at 4 degrees, would work. But, I need actual publication quality pics and I'm no where close. And, unfortunately I'm a grad student with not a whole lot of experience with antibodies. Thanks!
fly ab stain problemsantibody tsa abc
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