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: [email protected] Lab location: Lerner Research Institute NE4-214 -----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of Casper Hempel Sent: Monday, March 02, 2009 1:53 PM To: [email protected] 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 _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet =================================== P Please consider the environment before printing this e-mail Cleveland Clinic is ranked one of the top hospitals in America by U.S. News & World Report (2008). Visit us online at http://www.clevelandclinic.org for a complete listing of our services, staff and locations. Confidentiality Note: This message is intended for use only by the individual or entity to which it is addressed and may contain information that is privileged, confidential, and exempt from disclosure under applicable law. If the reader of this message is not the intended recipient or the employee or agent responsible for delivering the message to the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please contact the sender immediately and destroy the material in its entirety, whether electronic or hard copy. Thank you. _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet
