RE: [Histonet] EDTA decalcification solution

From:"Tony Henwood"

Try this:

EDTA Decalcification Mixtures

Ethylenediaminetetraacetic Acid (EDTA) is a calcium absorbent that has
gained popularity because of its gentleness on tissue and antigens. It
is a slow decalcifier but the rate of decalcification can be increased
with use of microwave energy (Sanderson et al 1995 Biotech & Histochem

1.	To 1 litre of distilled water add 90ml concentrated ammonium
2.	Stirring continuously, slowly add 140g EDTA
3.	Adjust to pH 7.1 using concentrated ammonia.

Adjustment of the pH is essential. If the pH is too low it works only as
a too-weak acid. If the pH is too high (above 8) decalcification is
accelerated but alkalinity can be damaging to tissues.

EDTA has also been combined with Hydrochloric acid resulting in a rapid
decalcifier. In this case it seems to act by combining with the calcium
as it is solubilized by the acid. This allows the newly freed acid to
remove more calcium:

Dissolve 20g EDTA (di-sodium salt) in 1860ml of distilled water, and
then add 140ml concentrated hydrochloric acid. A precipitate may form
within 24 hours of preparation of the solution but this does not appear
to affect the usefulness of the solution.

Since EDTA will precipitate in alcohols, it is important to wash tissues
in water.


Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318

-----Original Message-----
[] On Behalf Of Fawn
Sent: Thursday, 19 October 2006 2:42 AM
Subject: [Histonet] EDTA decalcification solution

Hi everybody,

I am getting ready to decalcify some mouse femurs for immuno staining.  
I know I have to use an EDTA decalcifier, but I do not know how to make 
it.  Does anybody have a protocol on this that they would be willing to 

Thanks in advance

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