[Histonet] HIER

[Della Speranza, Vinnie]

You will definitely have to optimize for each antigen.

The time your slides are in your retrieval buffer is inversely related to the temperature.

The higher the temperature of your buffer, the shorter the time needed for retrieval. 
 
There is nothing wrong with retrieving at a lower temperature (lower than boiling) however you will have to significantly increase the time in order to attain a satisfactory result. If time is not an issue for you, you may be able to retrieve at 90-95 degrees (below boiling to avoid boiling off your buffer) but you may have to double or even triple the time your slides are left in the retrieval buffer at that lower temperature.

Vinnie Della Speranza
Manager for Anatomic Pathology Services
Medical University of South Carolina
165 Ashley Avenue  Suite 309
Charleston, South Carolina 29425
Tel: (843) 792-6353
Fax: (843) 792-8974
 
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Casper Hempel
Sent: Tuesday, March 03, 2009 4:13 PM
To: rjbuesa <@t> yahoo.com
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] HIER

Thanks for all your useful comments. It sounds like to choose temperature
over pH depends on the epitope in question more than anything else. We'll
probably have to optimize it for each antigen.
Best regards
Casper

2009/3/2 Rene J Buesa <rjbuesa <@t> yahoo.com>

> 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
> _______________________________________________
> Histonet mailing listHistonet <@t> lists.utsouthwestern.eduhttp://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet