[Histonet] EDTA

[Tony Henwood]

José,

I have inserted my ideas below within your email.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Jose Luis Palazon Fernandez
Sent: Monday, 10 October 2011 10:34 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] EDTA

 
Dear List members

Greetings
> I would like to ask you some questions about the use of EDTA as demineratizing agent.
> I have to do histology and histochemistry (including metallothioneins) in fish larvae preserved in Bouins solution.

How long do you fix in Bouins solution? Bouins, being an acidic fixative (picric and ascetic acids) will fix as well as decalcify, especially fish larvae which would have little calcium compared to human trephine specimens. So I am not sure whether you will need to decalcify.

> 1) First of all I would like to know what is the best formula (or the one you recomend) to prepare the EDTA solution, as well as the importance of maintaining a given pH and if it is necessary to put the > > samples in the refrigerator during the demineralization process.

The following solution was used by Sanderson et al (Biotech & Histochem 70(1):12-18, 1995):

1.	To 1 litre of distilled water add 90ml concentrated ammonium hydroxide.
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.

> 2) Is EDTA recomended only for certain fixatives, or It can be used with any fixative, including Bouins

Again why do you need to decalcify these specimens, since Bouins will also decalcify

> 3) When using Bouins solution as fixative, many books recommend not to wash in water but instaed to remove the excess of picric acid using several washes in etanol 70%, on the other hand, I´ve read > that EDTA precipitates in the presence of alcohols. What do you recomend in this case?

I also recommend washing in 70% ethanol since protein picrates are believed to be water soluble. I would suspect placing the Bouins fixed tissues in an aqueous EDTA solution would do the same thing (ie allow extraction of the picrates).

> 4) Do you think that the demineralization procedure could alter or affect the detection of metallothioneins in the tissues?

It would be possible that the Bouin's fixative might affect the antigens recognised by your metallothionein antibodies but unlikely that EDTA treatment would - only by testing would it become clearer. Are your antigens affected by Bouins?

Many thanks in advance
Best regards

José Luis

Dr. José Luis Palazón Fernández
Instituto de Investigaciones Científicas Universidad de Oriente Boca del Rio-Isla Margarita-Venezuela Direccion actual: Instituto de Ciencias Marinas de Andalucia-CSIC Campus universitario Rio San Pedro, 11510, Puerto Real, Cádiz, España
email: jluis.palazon <@t> icman.csic.es; jose.palazon <@t> ne.udo.edu.ve
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