[Histonet] Re: 14% EDTA

[gayle callis]

There is no vendor I know of who sells 14% EDTA.  It is important to
remember that the EDTA you have to use is Tetrasodium EDTA, MW 452.2.   This
particular EDTA is highly soluble in water compared to EDTA without any
sodiums, or disodium EDTA.  Also, once dissolved it has a very high pH of
approximately 9 or more and you must adjust the pH DOWN to pH 7.2 - 7.4,
although we use pH 7.6 without problems.  If you do not adjust the pH down,
you will damage alkaline sensitive protein linkages in your tissues/bone.
Do not use HCl to adjust the pH, but glacial acetic acid.   The publication
supporting this EDTA decalcification was written by Dr. Webb Jee many years
ago.  We prefer to use tetrasodium EDTA because we can achieve a high
concentration with the highly soluble EDTA. 

 

You can buy tetrasodium EDTA if from any vendor, ThermoScientific, VWR, etc.


 

Simple recipe is

 

14 g Tetrasodium EDTA mw 452.2 (this may vary) 

80 ml Dulbeccos PBS or water (the latter is fine IF your bones are totally
fixed)

Adjust pH DOWN to 7.4 with glacial acetic acid (it takes a fairly large
volume)

Bring final volume to 100 ml with either buffer or water, depending on what
you dissolved the salts in. 

 

We adjust pH using constant stirring and pH electrode in the stock solution
to watch the pH change.  Keep track of final volume of glacial acetic acid,
so the next time you make this up, you can add a large volume without having
to keep adding glacial acetic acid with a pipette (in sense, a titration to
correct pH). 

 

As with all EDTA decalcification, it takes longer and you must rinse the
bones well for a couple of hours or the EDTA will ppt out in alcohols,
creating some annoying sectioning problems.   

 

Suspend your bones in this solution and use endpoint testing to know when
calcium is removed.  We use a simple weight loss weight gain endpoint test
that works very well for EDTA unless you have an xray machine or microCT
scanner to detect Ca removal.  EDTA is not affected by temperature, and can
be performed in the cold although room temperature works perfectly.  

 

Gayle M. Callis

HTL/HT/MT(ASCP)