Gayle,

Could you tell me more about IMEB. I make my own 10% EDTA and we use a ton
of it. I am interested in finding some place to make it to my
specifications. 

Thanks 

Jennifer Harvey
University of Rochester 
Department of Ortho/Path
Rochester NY
716-273-4129

> ----------
> From: 	Gayle Callis
> Sent: 	Monday, December 18, 2000 8:55 AM
> To: 	HOOVER_JENNIFER@LILLY.COM; ANATECH LTD; histonet@pathology.swmed.edu
> Subject: 	Re: EDTA as Decalcifying Agent?
> 
> 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
> 
> >question  
> 
> 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 
> >debate.  
> 
> 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
> 
>