Co-staining Protocol - What's wrong here? (Oct/22/2009 )

So I've been learning how to do immunostaining, but the only protocol that's been successful was the one I used for just tyrosine hydroxylase (1:500 rabbit anti-TH, 1:200 Alexa Fluor goat anti-rabbit 488, 3% NGS). I incubated with NGS and 0.3% Triton before the primary, which was also in 3% NGS. I also did a Hoechst stain. Everything was good.

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!

I think the problem was in NGS but not NDS.
The use of normal sera as blockers requires a matching of the animal species to the secondary Abs. For your anti-TH staining, NGS matched goat-anti rabbit conjugate and did not interfere the rabbit primary Ab. However the presence of NGS in the co-staining protocol would prevent detection of the sheep anti-ChAT, because goat and sheep IgG are highly homologous.
Instead of trying new combinations of primary and secondary Abs and sera to match them, I would recommend to replace both NGS and NDS with a serum-free protein blocker, e.g. casein solutions etc.

Hey, thanks for the response. You make a good point, but I would also point out that two attempts at single staining for ChAT with NDS failed as well. Any thoughts on that issue?

Has your anti-Chat antibody been validated for IF? (Not all antibodies will work in IF)

If it has been validated in IF, how were the cells fixed (methanol,acetone, PFA)? Have you tried different fixatives? An antibody may require a particular fixation method (ie the epitope may be destroyed by a certain type of fixation)

How long are you staining with your primary? Have you tried overnight?

Have you validated that your secondary antibody is working by testing it with a different primary?

How were the cells permeabilized? Have you tried 0.1% saponin? methanol? acetone?

Does the primary work for westerns or some other application that you could use to test the ab?

miBunny on Oct 27 2009, 08:40 PM said:

Hey, thanks for your help. IHC is listed by Millipore among the applications of the antibody. There are also papers that have used it successfully with protocols similar to mine. I fix the tissue with 4% PFA, as do all of the papers I've read on ChAT staining, so I'm assuming there's no epitope destruction. I stain with my primary at 4 degrees overnight. The secondary has not been proven to work with a different primary, but we don't have a working primary to test it on. Any ideas? I use 0.3% Triton to permeabilize, which is published in papers with my ChAT anitbody and others, but might try Tween as well. When I co-stained with TH, the TH staining worked fine, by the way, which leads me to believe permeability was not an issue.

Thanks so much for your help again. I'll be doing some diagnostic staining in the next day or two with different concentrations and incubation methods and will post updates.