[Histonet] EDTA

[Jose Luis Palazon Fernandez]

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.

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.

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

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?

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

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
tlf: +34-956832612; fax: +34-956834701; cell: +34-600487100