[Histonet] pressure cookers for IHC

[Pritchard, Michele]

Hi Jennifer:

I wrote this response to Casper Hempel 03 March 2009 when he posted a question regarding the relative importance of temperature and pH in HIER techniques to the Histonet community.  I copied and pasted it below (and edited it for grammar, organization and spelling this time!) for your information.


I have a reproducibly good method for HIER 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 heating process. 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. 

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 occurred 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 Jennifer Campbell
Sent: Monday, March 30, 2009 10:44 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] pressure cookers for IHC

Hi everyone,
 
Does anyone use a pressure cooker for heat induced epitope retrieval?  I
know there are a lot of fancy antigen retrieval units out there that you
can buy specifically for IHC but, I know a lot of people just use a
pressure cooker with the same success.  Any specifics as to what type of
pressure cooker to use? Protocol used as far as the temp and for how
much time?  Any info would be appreciated.
 
Thanks,
 
Jen C.
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