[Histonet] HIER

[Pritchard, Michele]

Casper:

I have a reproducibly good method for HEIR for two Abs I routinely use (alpha smooth muscle actin in mouse liver and proliferating cell nuclear antigen (PCNA) in mouse liver).  

I use a microwave pressure cooker (Nordic Ware, tender cooker).  Into the pressure cooker, I place 300mL of distilled water to keep the humidity high during the in my pressure cooker. I use a citrate buffer solution which contains 0.01M sodium citrate dihydrate and 0.04M citric acid monohydrate, pH 6.0.  I place 500mL of this solution into a 1.5pt (710mL) 'servin' saver' container and set the filled container into the pressure cooker (into the distilled water).  I slip my slides into an autostainer rack and place the rack on its side in the citrate buffer soln.  I microwave the entire setup at 50% power (1100 watt microwave) for 20 minutes and then allow the cooker to cool down for 20 additional minutes.  

When I remove the cooker from the microwave, I open it on my bench and take the temperature of the citrate buffer.  It is usually between 95-98C.  After the 20 minute cool-down, the temperature of the citrate buffer is around 56-57C. Once completely cool, I test the pH of the citrate buffer to ensure that it is still at pH 6.0.  (I have taken these measurements a total of 11 times.)

My single failure when using this method of HIER was when the temperature did not get hot enough. I was rather naïve at the time and thought I would proceed with the staining and see what happened, assuming the pH was more important than the temperature.  So much for that hypothesis.  

Mind you, I fully disclose that my experience is rather limited to the two staining protocols I have optimized, but I offer up that experience for your edification.  Hope it helps!

Kind regards:

---mtp

Michele T. Pritchard, Ph.D.
Research Associate
Nagy Laboratory
Department of Pathobiology/NE40
Lerner Research Institute
Cleveland Clinic
9500 Euclid Avenue
Cleveland, OH 44195
 
phone:  216.444.8613
fax:  216.636.1493
 
email:  pritchm <@t> ccf.org
 
Lab location:
Lerner Research Institute
NE4-214
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Casper Hempel
Sent: Monday, March 02, 2009 1:53 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] HIER

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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