[Histonet] Thionin staining method

[Runyan, Caroline (NIH/NIMH)]

I am trying to re-stain brain sections with thionin (they had been stained
four years ago initially but were originally very light and have now faded
beyond recognition).  I popped off the coverslips, and then left the
sections to sit in xylenes for about five days, at which point the dpx used
for coverslipping appeared to have been removed from the tissue.  Then, I
re-hydrated and incubated in thionin for fifteen minutes-the thionin did not
stick to the tissue at all.  As soon as I drained the slides in water, all
color was removed.  I then went back and looked at the protocol that was
apparently used to stain the tissue originally, and it looks as though the
tissue was not de-fatted prior to thionin.  The slides went from 70% EtOH
(no water or 50%) through to 100%, into thionin, and then straight to 95%,
100%, xylene.  
 
Is tissue's current lack of thionin reactivity due to the fact that the
original stain had been done so long ago, or because of the procedure
originally used?  Thanks.