I just completed a whopping big project of adult trout heads (looking
for whirling disease) which EDTA decal was necessary.  Below is my
method for preparing 1 liter of EDTA decal solution.

I.  Prepare a PBS solution.  I used Na phosphates.  I cannot vouch for
how K phosphates will perform in EDTA.  
	dibasic.... 6g/liter DI water
	monobasic.... 4.5g/liter DI water
	NaCl ........ 8.1g/liter DI water
   
In approx. 700 mL of PBS, add 100 g EDTA and begin stirring. Bring pH up
to 7.2-7.4 by adding pellets of NaOH (this may take aprox. 5 g or more
to do).  Use either a pH meter or litmus paper to determine the pH.  I
used litmus paper.  After the EDTA is COMPLETELY dissolved, add DI water
qs to 1 liter.  If you overshoot the pH (more alkaline that 7.40),
adjust with HCl. If you use conc HCL, you can do it with just about 1 or
2 drops (the voice of experience).  Try to NOT overshoot the pH, but if
you do, minimize the amount of HCl you need to use. If you overshoot big
time, just add more EDTA/PBS to the mix and go from there. I made my
solution up in huge quatities (2 x 5 gallon containers full), so I just
dumped the NaOH in by the spoonfulls (each spoonfull weighs 11.5g), but
if you're just making up 1 or 2 liters, be carefull with the NaOH.  

This whirling disease project was the first time I had ever used EDTA as
a decal solution and I found that i really like it.  The tissues take a
bit longer to decal, but the soft tissues are preserved much nicer than
my regular decal solution and they also cut softer.  I also found that
for those tissues that have really hard crunchies in them (like a kidney
with kidney stones), soaking in EDTA decal solution for about 1-2 hours
really softens them up and they cut like butter. I love this stuff.

Hope this helps.

Connie McManus




jane weeks wrote:
> 
> Histonetter's -
>         Does anyone have a protocol for making EDTA - based decalcifying solution?
> 
> Thanks!
> Jane Weeks
> McArdle Lab for Cancer Research - UW
> Madison, WI

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