I am doing IHC for normal proteins, but there must be a different protocol to treat phospho proteins. Please, enlighten me with that.
Thanks!
In my experience, correct perfusion with a fixative (e.g. PFA) should be enough to maintain the phosphorylated proteins in place.
But you might want to add phosphatase inhibitors to the saline solution (prior to PFA flush), for example Sodium Fluoride. And maintain the inhibitor in all the buffers used afterwards.
I never had better results doing that, but some P-proteins should need it, specially if they are low abundant.
https://www.neb.com/products/p0759-sodium-fluoride-fluoride
We have a nice IHC method, attached.
BSA V im told is a big deal. Expensive but great product, many labs use it.
Also, sodium borohydride. people talk about the glycine, as a great additive. But borohydride give the phospho proteins IHC a kick.
Remember, about the monoclonal antibody, start soft, then go harsh. For activity dependent proteins, you might need to stimulate tissue beforehand. Do not run trials on old tissues (depending on antibody - 1.5 years ?).
You use the phase "but there must be" in IHC, every epitope, every antibody, every tissue is different. Take good notes, no trial should ever be, "worked or didn't work" every trial holds a secret. Often, to much Triton-X, or use Tris buffer saline instead of PBS. How are the pH of your solutions, do they ever change drastically? Sometimes you dont have to do anything different. Include controls, can you run a Western to know if actually present? Look out for simply dots when doing microscopy, that might actually be your protein- or not.
If all else fails, make fresh buffers and solutions. Specially if having issues in combined antigen retrieval protocols.
Do not use images from manufacturer website to compare with your results. Only use published articles.
Sorry saturating answer, IHC, like art, can be frustrating but certainly rewarding.
Links attached,
http://www.pnas.org/content/108/2/840?ijkey=b2eaec24db1f8bd8a2faa78663fb3a3cd1aace99&keytype2=tf_ipsecsha
http://www.jneurosci.org/content/36/19/5228.long#sec-2
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