[Histonet] IHC in Fixed Frozen vs. Processed and Paraffin Embedded Brain Tissue

[CM Bush]

Hello there Dear Histonet,
 
I am re posting this from a few days ago.  The were no responses, so I'm hoping maybe this time.  Much thanks for everyone's patience.

We are trying to stain human anterior cingulate brain with anti 
Calretinin.  We have taken a sample of said tissue (fixed in 10% NBF);
processed and paraffin embedded one portion of the tissue and cut the other 
portion on a freezing stage microtome (fixed frozen sample cryo protected 
with 30% sucrose).   (The sections for both varieties mounted on slides.) 

We have been able to see Calretinin staining (at 1:3000)  for the 
processed, embedded tissue but not for the fixed frozen version of the same 
tissue.  For all tissue, antigen retrieval was done using citrate 
buffer pH6 and microwaving 12 min high and 6 min low, in a pressure cooker. 
Also, we used  TBS buffer with 0.4% Triton X for all sections. 

Would anyone have any ideas as to what might be going on that the 
processed/embedded tissue will stain and the fixed frozen version of the 
same sample does not? 

Thank you very much, again.
 
Clarissa