Re: antigen retrieval with EDTA

From:"J. A. Kiernan" <>

On Wed, 28 Mar 2001, Don and Cathy Schaefer wrote:

> Does anyone have a recipe for EDTA (ph8.0) antigen retrieval solution?

Here are 2 references. Both used 1.0 mM EDTA adjusted to pH 8.

Balaton AJ, Vaury P, Baviera EE, Vuong PN, Galet BA: Protocole 
  "EDTA-autocuiseur." Une technique immunohistochimique 
   performante. Ann Pathol 15: 295, 1995

Pileri SA, Roncador G, Ceccarelli C, Piccioli M, Briskomatis A,
  Sabattini E, Ascani S, Santini D, Piccaluga PP, Leone O, 
  Damiani S, Ercolessi C, Sandri F, Pieri F, Leoncini L, 
  Falini B: Antigen retrieval techniques in immunohistochemistry: 
  Comparison of different methods. J Pathol 183:116-123, 1997

The second paper was a major comparative study of different
antigen retrieval methods for several antigens. For a majority
of antigens EDTA was superior to the more frequently used pH 6
citrate-citric acid buffer.

To make the solution it doesn't matter whether you use EDTA
itself (the acid) or one of its sodium salts. (You should not
use a calcium EDTA because one hypothesis about antigen
retrieval is that it works by removing calcium ions. Ask me
for references if you want to follow up.) 

The generally most useful and widely used salt is crystalline 
disodium EDTA (Na2[EDTA].2H2O), M.W. 354. To make a 1 mM
solution dissolve 0.35 G in 1000 ml of water. Add drops of
1 M sodium hydroxide (that's 4%) to adjust the pH to 8. If
you overshoot, correct with 1 M HCl (a twelve-fold dilution
of conc. hydrochloric acid). 

This and the next 2 paragraphs have nothing to do with 
antigen retrieval, so if you're a dedicated immunohistochemist
you can stop here and you'll miss nothing that could
possibly make you better at your job.
Disodium EDTA is useful stuff to have around. If you live
in a place with hard water and your kettle gets scaled up,
a teasponful of EDTA in a kettleful of water gets rid of
it very efficiently. Boil for 10 minutes, leave it for a
few hours, then wash out with a good blast from the tap
to get rid of any unchelated chunks. It works well in
lab water baths, too. It doesn't shift black stains
(manganese dioxide). For these you need a teaspoonful
of either oxalic acid (odourless) or a bisulphite (rather
acrid), swished around in 200 ml or so of warm water.

We who work in labs have even less excuse for encrusted
kettles than we have for that other great scourge of the
Western World, known as ring-around-the-collar, for 
which the only known remedies are: [a] washing
and (if courageous and fastidious enough) shaving the
back of the neck, [b] obeying the telly - buying and
generously using an expensive mixture of detergent and 
bleach - or [c] scrubbing the dirty bits with a little
brush before chucking the shirt in the washing machine.
EDTA does nothing for collars.

John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1

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