Then I attempted to co-stain for TH and choline acetyltransferase, using the same protocol for TH but adding the necessaries for ChAT (1:1000 sheep anti-ChAT, 1:200 Alexa Fluor donkey anti-sheep 594). I used 3% NGS and 10% NDS in my blocking and primary solutions. I found TH, but no ChAT staining. I've also attempted to co-stain with an anti-GFP antibody and an anti-c-Fos antibody, and in that case I found little or no staining for either target.
My next troubleshooting step was to stain for just ChAT, but I saw no staining in that case as well (was looking in the PPTG, LDTG, and the ventral PAG). The primary could be bad, but I have yet to run a test to establish that. Is there something obvious I'm missing here? It seems as if all of my problems began when I started using NDS. Would it be a good idea to try some new NDS? This stuff may be old.
I appreciate any help, and let me know if I left anything important out. Thanks!
Edited by kegallagher, 22 October 2009 - 03:48 PM.