[Histonet] Re: [IHCRG] HIER

[Kim Merriam]

In my experience, you can use the Biocare Decloaker with their default program for most protocols (I believe it heats @ 125C for 10 seconds and then 90C for 10 minutes, but dont quote me on that because I am not sure).  I do not preheat my buffer when I use the pressure cooker, since it heats up pretty quickly.  I have used a lower-temp protocol for some phospho-proteins (90C for 30 minutes), but that is the exception and not the rule.

I also use a Black & Decker steamer on a regular basis.  I always preheat the buffer (in the microwave) before putting it in the steamer, because the steamer heats slowly and does not get to temps that are as high as the pressure cooker (I check the steamer regularly and it gets to about 100C or so on most days).  I usually steam the slides for 45-60 minutes.

In the case of both heating systems, I usually let the slides cool for about 10 minutes and then rinse them in running water.  I have heard of other people cooling their slides for up to an hour and still others that do not cool at all.  I am not sure that how long you cool the slides really has an impact on anything (IMHO, it is all about the heating and not the cooling).

As far as pH is concerned, that a whole other topic.  For most antibodies, a routine citrate or EDTA buffer works fine (pH 6-8 or so), but I have used the high-pH buffers on occasion (usually for nuclear proteins).

I work in a biotech/ discovery research lab, so my particular lab does not need to follow strick regulatory guidelines (although I try to run the lab in a "GLP-like" fashion).

Just my 2-cents.
Kim
 Kim Merriam, MA, HT(ASCP)QIHC
Cambridge, MA 




________________________________
From: "Perry, Margaret" <Margaret.Perry <@t> sdstate.edu>
To: "ihcrg <@t> googlegroups.com" <ihcrg <@t> googlegroups.com>; "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>
Sent: Wednesday, October 22, 2008 12:28:24 PM
Subject: [IHCRG] HIER


We have been using the microwave HIER and have had good results, however when our microwave bits the dust I would like to have a pressure cooker method in place.  I feel the pressure cooker is more consistent for all the slides.  We are a veterinary diagnostic lab and I would like to have some idea of where to begin.  I have looked at different protocols and they often indicate HIER in a pressure cooker but do not give the details.  I currently use citrate buffer pH 6.  I put the slides in refrigerated buffer and microwave on high for 1 min 45 sec. or until the buffer just starts to boil.  I then set the microwave on 10% power for 10 minutes.  Afterward the slides are allowed to cool in the microwave for 1 hour.  We have a biocare Decloaker Chamber and I would appreciate help with the program I should use.  Do you start with cold buffer or should I prewarm it?  What temperature should I use? How long should I maintain the temperature?  How
 long should it be before I remove the slides?
 
I also am working with a new protocol that calls for heating in a steamer.  Should the temperature of the buffer be warm, cold or room temp when I start?
 
Thank you.
 
Margaret Perry HT (ASCP)
IHC Lab Manager Veterinary Science
Animal Disease Research and Diagnostic Lab
South DakotaState University
Box 2175 North Campus Drive
BrookingsSD 57007
 
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