The inquiry about old bones stored in formalin for 5 years.  Unless the pH
of the formalin was maintained at pH 7, i.e. changed to maintain fresh NBF,
you may encounte problems. Formalin tends to break down to formic acid over
time, the pH of "NBF" becomes acidic, and bone can also become a tidge
decalcified. We had bovine bones stored for many years without changing
NBF, and when trying to do undecalcified PMMA method, the bone literally
twisted apart in the plastic block.  We should have stored the bone in 70%
alcohol IF we wanted undecalcified bone.   

If NBF has been allowed to drop below 6.8 or lower, nuclear staining may
also be damaged by this lower pH. You can try so see what happens, but be
prepared for some changes.  Heavy duty cortical bone may withstand storage
better than trabecular bone and soft tissues.  You might have to adjust
your staining to have optimal results.  

I think I would decalcify in formic acid rather than HCl for your old
bones, but you may get lucky, worth a try. 


Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu