Dear Robert,
I don't think that there is an endpoint in NBF fixation. To my opinion the cross-linking will go on and on. We had a heart collection in NBF dating back to 1970. From time to time there was a IHC request on those old samples. Well, before the description of antigen retrieval (1992) it was fully impossible to stain anything except for some weak staining with anti-von Willebrand factor (after pepsin treatment of the sections). With antigen retrieval, it appeared that it was fully antibody dependent whether or not reliable staining was obtained. Also when there was a request of a series of specimens ranging from 1970 up to recent years, there was an increasing staining intensity of that antibody. Most successful was anti-alpha actin, clone 1A4 for staining smooth muscle cells. I think that one should be able to stain SMC's in mummy tissue! But detection of T-cells by CD3, CD8, etc. was totally impossible in those old specimens. However, the introduction of an ultra sensitive de
tection system based on tyramide amplification definitely improved the situation. CD3, CD8 can be detected now by tyramide visualization in 10-20 year old specimens.
  
I agree with you there is not much literature about this subject. Hope this opinion helps.
Chris van der Loos, PhD
Dept. of Pathology
Academical Medical Center
Amsterdam - The Netherlands 

----- Original Message ----- 
>From  Robert Krug  
Date  Wed, 22 Sep 2004 13:57:28 -0500 
To  histonet@lists.utsouthwestern.edu 
Subject  [Histonet] Long term storage of tissue 
I have a question concerning the long term storage of tissue which is not 
addressed in any of the histology textbooks I have on hand.  After many 
hours of searching, I was unable to find an  answer through a websearch.

Can tissue be preserved in 10% neutral buffered formalin for decades 
without changing the 10% NBF (provided the container is sealed and does 
not leak)?

A former coworker of mine once worked at the CDC and she stated the CDC 
used 70% alcohol  for the long term storage of tissue.  A review of the 
Internet shows that the Australian Museum Fish site states they have fixed 
in "formaldehyde and then stored in alcohol"  for the last 80 years.  This 
site claims to have thousands of specimens on hand.    Another site I 
found recommended storage in formalin as a way to keep the original color 
of the tissue and recommended against long term storage of tissue in 
alcohol for various reasons. 

The unused 10% neutral buffered formalin solution does decompose over time 
and is typically assigned an expiration period.  Once the tissue has been 
fixed in 10% neutral buffered formalin, is the reaction stable, meaning 
the bonds are stable and no decomposition will occur?  I was always  told 
the formalin solution degrades with time and may form formic acid and 
other by products.   Given enough time the remaining formalin in solution 
might  form formic acid, drop out of solution as paraformaldehyde or 
escape as formaldehyde gas.   Another coworker feels that once fixation 
has occurred, the fixation is permanent and there is no need to change the 
solution.  I realize the fixation bonds can be broken through washing in 
running water and with select chemicals.  But given a sealed container, 
will the bonds remain permanent and the tissue free of decomposition or 
fungal growth?

Any jounal articles or textbook references you have to support your view 
point would be greatly appreciated. 

Many thanks

Bob Krug
St John, Missouri



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