I have been using fluoro-jade B on two types of brain tissue- fresh frozen and that perfused with 4% PFA, post-fixed with PFA for 24h, incubated in 30% sucrose for 72h then stored at -20C before being cut on a cryostat. The stain works nicely on fresh frozen tissue, but in PFA fixed tissue only gives dim staining, not easily differentiated from control staining on the contra-lateral side. I have tried omitting the pot perm stage to try and increase staining (including background) but this doesn't make much difference. Has anyone else had this problem and does anyone have any ideas how to fix it,
   
  thanks,
   
  Lisa

       
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