Hi there,

To overcome the lost of antigenicity produced by routine fixation techniques you can search for "Antigen retrieval". There is a wide variety of these techniques that are supposed to break the cross-links in between the proteins produced by formalin fixation, thus allowing antibody detection of specific epitopes.

Reference: Article Molecular mechanisms of antigen retrieval: Antigen retrieval...

In the link below you will find summarized some of these protocols. However, I'd advice to look in the literature for specific Ag retrieval techniques performed on the tissue and fixation conditions you are working on and that have shown success unmasking the antigenicity of your protein of interest. I am saying this because different Ag retrieval techniques could render different results with different antigens...

http://www.ihcworld.com/epitope_retrieval.htm

Cheers,

J

Prolonged fixation destroys the antigenicity. Due to this it is not advisable to fixate your tissues for so long. Even without prolonged fixation you still need to retrieve your antigens with heat or enzymes.

It depends of the tissue sice of the tissue whether you can talk about an overfixation. If you like to fix a rat brain by immersion fixation you need 4 -6 days. at first try your antibodies and look whetehr you see anything. In that case you should use a positive control to compare the result. If you are using paraffin sections then antigene retrieval is recommanded to improve your results. If you are working with frozen or Kryosections it's better to work with higher antibody concentrations, longer reactions times, instead of overnight rection , 4 days in the fridges, or more sensitive detection systems, instead of labeled secondary antibody use biotinylated secondaries and labeled Streptavidin as a detection system. Finally it depends on the primary antibody you like to use. Good luck!

Dear all. Thank you gentlemen for your helpful insights and suggestions, especially the reply from Dr. J. Ramirez-Franco.