Another Re: [Histonet] Bone decalcification

From:Gayle Callis

Acid decalcification BEFORE fixation will macerate your tissues aka protein 
hydrolysis.  It will destroy both soft tissue morphology and nuclear 
staining, and there is no way you can repair this kind of chemical 
destruction of tissues.

Total fixation is needed to protect the tissues from the effects of acid 

IF the bone is small, i.e. a bone biopsy or cut down into a very small 
piece,  then a formic acid/formalin (combination fixation and 
decalcification) can be used successfully.  This is NOT recommended for 
really large bones (mfrs even make these recommendations in their 
instruction sheets).   One message kindly provided vendor recommendation 
for this fix/decal solution.

If you do not want to use formic acid/formalin, then fix the bone first in 
NBF and proceed to a acid decalcifier.  Keep in mind, strong acids i.e. HCl 
(commercial solutions work very well) can damage antigens if bone is left 
in these solutions too long.  For a gentler acid decalcifier, buffered 
formic acid is available commericially.

The recommendation about a textbook is a good one - several devote whole 
chapters to bone histotechnics, including how to decalcify, etc and what 
will happen IF you do not perform these procedures carefully.

Our personal preference is to totally fix the bone in NBF.  To speed up 
both fixation and decalcification, reduce the size of the bone i.e. cut a 
representative sample for fixation followed by decalcification.   This also 
helps with the combo fix/decal method.  If you suspend the bone, the 
reagents surround all sides of the bones.

The only time I have seen unfixed bone followed by decalcification is with 
EDTA, and it was a tedious, long drawn out method, used for research 
projects and NOT the clinical setting where you need a diagnosis asap.  The 
EDTA methods are found in several publicaitons.

At 04:40 AM 4/24/2006, you wrote:
>   I just wanted to know other histo techs opinions on bone decalcification.
>I learned in school that you decal the bone than you fix it. I have a
>pathologist who claims she learned it the hard way, and that it is better to
>fix the bone than decal it. What did you learn on how to do this procedure?
>This pathologist claims that fixing prior to decaling keeps the cells more
>intact. Any opinion on your procedure or your technique is appreciated.
>Heather A. Harper
>Naval Hospital
>Pensacola, FL
>Histonet mailing list

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

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