Re: EDTA as Decalcifying Agent?

From:Gayle Callis <>

At 01:27 PM 12/18/00 -0500, you wrote:
>        Hello Histonetters,
>                I have  a few questions regarding EDTA as a decalcifying 
>agent.  1)  why is it not more commercialized (ready-made) which leads to 

You can buy it commercially prepared, Poly Scientific, Newcomer Supply,
Rowley Biochemical, probably Decal Corp have availability.  I think IMEB
will custom produce a decalcifier to your specifications.  

2)  when I prepare EDTA in the laboratory what is the 
>approximate shelf life?  

MSDS may help here, I think it is very stable in water, with PBS, something
might grow?? but as in all things, expiration dates can, will and should be
given, you would probably use it up before expiration date anyway.  

I have read my histology books and found 
>reference to EDTA but I know the veteran histonetters and bone researchers 
>are probably proficient with knowledge on this particular subject.  Also, 
>are there any good reference articles out there dealing with decalcifying 
>agents and effects on tissue?  

Sorry folks! Tooting horn a tidge, Callis and Sterchi, Decalcification of
bone;  literature review and practical study of various decalcifying
agents, methods and their effects on bone histology  J of Histotechnology,
21(1):49-58, 1998 with extensive referencing on effects, IHC and
proteoglycan work, etc.  Happy to send original reprint. 

I also need some feedback on how important 
>is it to rinse the tissue in running tap water after acid decalcification 
>to remove acid residue before processing?  This has been a topic of 

Rinse! I tried debating it, and decided rinse was a better choice,
particularly large bones.   Usually an hour to 4 hours, bone slabs, 3 - 5
mm thick, smaller- less time, and with EDTA at must, or a ppt forms when it
comes in contact with alcohol, crunchy sectioning (Yuck!) 

 The main thing is to STOP decalcification, rinsing helps do just that,
then into 70% alcohol (Culling recommended 2 or 3 changes of alcohol rinses
to get rid of acid, and the alcohol stops the decalcification action).
Long overnight rinses can create swelling, however, residual acid will
continue protein hydrolysis after calcium removal, leading  to ROTTEN
nuclear/soft tissue staining/damage.   Better to err with some swelling
than ruin precious staining characteristics and antigens.  You can
neutralize acid with several changes of saturated lithium carbonate or
sodium bicarbonate, test with litmus paper to neutrality, rinsing is
cheaper and easy.  Small bone biopsies are often plunked into processor
without problems. Large or numerous bones, acid decalcified contaminate
reagents, be prepared to change these more often if you don't rinse or risk
ruining other processed tissues.  Debated for years, lost arguement and
rinse faithfully. 

Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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