[Histonet] HIER

[Rene J Buesa]

Both temperature and pH go hand in hand and it is very difficult, if not impossible, to select one over the other without experimental data.
The thing is to just boil the container with the pH buffer and once it has boiled (usually in 20 minutes) take it out and leave it on the counter for another 20 minutes.
The pH (from 6 to 8) will depend on the epitope that is going to be retrieve because some need pH6 and other pH8 and even higher (or lower). Seldom pH7 (neutral) is used.
René J.

--- On Mon, 3/2/09, Casper Hempel <casperhempel <@t> gmail.com> wrote:

From: Casper Hempel <casperhempel <@t> gmail.com>
Subject: [Histonet] HIER
To: histonet <@t> lists.utsouthwestern.edu
Date: Monday, March 2, 2009, 1:52 PM

Hi histonetters
We have been talking a lot about how to retrieve your epitopes using a
microwave in the best possible way.
We haven't come to an agreement and people in my lab both argue that
temperature and pH are important issues. Without a doubt both factors are
important for a proper retrieval, but if you have to focus on one of the
factors, which would you consider the most important? Temperature or pH?
The issue is mainly longer incubations of the slides in boiling buffer. The
buffer is evaporating and the solution/buffer gets less and less pH neutral
and you need to top up with dH2O. However, if you boil with reduced
intensity, less evaporation will occur and the pH will remain more stable.
Do you have any suggestions or comments to this issue?
Looking forward to your replies.
Cheers
Casper
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