RE: IP's with Decal

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From:Gayle Callis <>
To:"Morken, Tim" <>,
Content-Type:text/plain; charset=us-ascii

How long EDTA takes to decalcify can depend on size, type of bone -
cortical bone takes longer than trabecular bone, species and age. 

EDTA is only soluble to a certain degree, usually around 10% for EDTA or
disodium EDTA, at pH 7.2-7.4.  IF you use tetrasodium EDTA, the pH will be
approx 8 - 9, and must be adjusted to pH 7.4 with ACETIC ACID, don't use
hydrochloric for this adjusment. 

EDTA decalcifies as a function of pH, this is the nature of the chelation
process with this molecule, and will not decalcify at pH 2 or 3, begins
around 4-5, increases at pH 7, with pH 8 optimal.  WARNING:  alkaline
sensitive protein linkages can be damaged, and EDTA is commonly used at pH
7- 7.4 or so, that is why 14% TETRASODIUM EDTA is adjusted to this pH
range.  Higher pH will decalcify faster, but you may do tissue damage.

EDTA will EXTRACT proteoglycans and is a biochemist's way of getting these
out of cartilage when doing chemical studies on these molecules. You should
stain a piece of normal, undecalcified articular cartilage in order to know
if you have loss of staining due to proteoglycan extraction with the EDTA. 

Our setup for cartilage studies with EDTA would be

1. Normal articular cartilage - undecalcified
   Normal articular cartilage - decalcified with EDTA
   These can be mounted on the same slide for optimal staining control,

2. Experimental bone/cartilage - decalcified with EDTA 

Large volumes help, heat can be used with EDTA ONLY if the bone is totally
fixed, or just use room temperature, suspend bone so all surfaces decalcify
evenly, gentle stirring is optional and not harmful, change EDTA solution
to replace used decalcifier several times with large bones (this is a
judgement call) and an endpoint check to know when calcium is removed is a
good idea - xray is excellent if available.

Good luck  

>I am using EDTA buffered to decal, too.  I made it myself to have a higher
>concentration of EDTA to decal faster.  Typically I'll have 12.% to 14% EDTA
>in the solution.  Boil your water in a microwave and pour EDTA powder in.
>That will dissolve EDTA very quickly.  Cool it down, then buffer it, and add
>other stuff.  You can have your 4% paraformaldehyde, or 10% NBF in the
>solution (if you fix your tissues using those) to fix and decal at the same
>time.  I also have some MgCl2 in my decal solution.  (Only add MgCl2 after
>the solution was buffered.)  This seems to help to keep my Safranin-O
>staining for proteoglycan good.  
>Other people decal at 37 degree, or using microwave.  This seems to speed up
>decal time.  But I have lost some antigenicity this way, so I prefer to do
>it at RT or at 4 degree with stirring.
>If you want to try my recipe, e-mail me.
>Good luck
>> -----Original Message-----
>> From:	Abizar Lakdawalla []
>> Sent:	Tuesday, August 22, 2000 1:07 PM
>> To:	Jennifer Englin
>> Cc:
>> Subject:	Re: IP's with Decal
>> EDTA (versene) based decal solutions work best for most immuno's.
>> Downside: it's expensive and takes much more longer to decal.
>> abizar
>> 1-877IGX-INFO
>> Jennifer Englin wrote:
>> > Has anyone found a decal that works well with immuno's?
>> > We are currently using Stephens Scientific Decal- formic
>> acid/formaldahyde/mathanol, and we are having trouble with our Kappa and
>> Lambda.
>> > Any suggestions?
>> >
>> > Jennifer,
>> > Willmar MN
Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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