[Histonet] Restoring nuclear staining to bone overexposed to acids, i.e. overdecalcified bone

Gayle Callis gcallis <@t> montana.edu
Wed Jul 13 14:07:11 CDT 2005


You can try placing the sections into 5% sodium bicarbonate for 10 minutes 
after deparaffinizing and rehydration then rinse well with running tap 
water, distilled then onto hematoxylin.

Also, stain longer in hematoxylin, even up to 10 min in Gill 2 or 3, 
Richard Allan hematoxylin 1, rinse with water and  IF you have to clarify, 
do only 5 dips to get rid of any hematoxylin on the glass slide, and 
blue.  If you use Harris, don't even use acid alcohol to differentiate, 
just stain 10 min, rinse and blue, rinse.  If you differentiate, you will 
take the minimal amount of staining away with the acid alcohol, been there, 
done that!!!   Some people have even stained overnight in hematoxylin to 
restore, but generally 10 minutes or so will work, hopefully.  See next 

Another possibility, and a long shot is expose the hydrated section to 1% 
periodic acid for 10 min, rinse and stain. This is an old trick for tissue 
samples stored for years in formalin that is degraded to a very acid pH.

Nuclear staining may be irretrievable as the acid, even formic acid, will 
have performed the little protein hydrolysis chemical number on the DNA, 
and you may never get good nuclear staining, and can't be restored.  If 
this is ongoing and using an HCl decalcifier, change to buffered formic 
acid, gentler, easier to control,  and perform decalcification endpoint 
testing.  Never leave the bones in decalcifier over a weekend, and if 
small, not overnight either.

Good luck

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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