[Histonet] Calretinin and 10% NBF Fixed Frozen vs. Processed and Paraffin Embedded Brain Tissue

[CM Bush]

Dear Histonet,
 
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 get 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 water filled, sealed secondary container. 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 won’t or hasn’t?  
 
Thank you very much, in advance.
CMB