[Histonet] elevation and HIER

From:"Liz Chlipala"

I'll chime in here also.  We currently use the pascal pressure cooker for
most of our HIER protocols, prior to purchasing the pascal we used a black
and decker steamer, we still use the black and decker steamer on occasion,
mostly for specimens which are a bit friable and have a tendency to fall off
the slide during retrieval.  But there are issues with the steamer.  In the
steamer and at my elevation I can only get the retrieval buffer to get to a
temperature of 93 to 93.5 degrees Celsius, that temp can work for some
immunos, but using the steamer can cause problems with consistency if you do
not monitor the temperature of your retrieval buffer through the entire
process.  I learned this the hard way.  It my opinion in order to use a
steamer at elevation and to maintain any consistency with respects to your
results you need to monitor temp through the entire process.  You can not
just start your steamer wait about 20 to 30 minutes and assume that its up
to the correct temp, put your slides and then time for 20 mintues, that's
not going to work consistently.  When we use the steamer we use a digital
probe thermometer that fits through the holes in the top of the steamer and
directly into the retrieval buffer.  Once the temp hits 93 + degrees we add
the slides and continue to monitor the temp, when the temp gets back up to
93 + degrees we start our timer.  That protocol seems to work well for the
steamer.   We much prefer using the pascal since its easy and does not take
that long as compared to the steamer which can take up to 1.5 to 2 hours to
complete the entire retrieval process. I have not used a microwave or water
bath at elevation so I can not comment on those methods.  


Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
P.O. Box 18592
Boulder, CO 80308
phone (303) 735-5001
fax (303) 735-3540
Ship to Address:
Premier Laboratory, LLC
University of Colorado at Boulder
MCDB, Room A3B40
Boulder, CO 80309

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Gayle Callis
Sent: Thursday, September 28, 2006 9:16 AM
To: Wulan Anggrahini; Histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] VCAM-1, ICAM-1, P-selectin antibody

We buy our antibodies from BD Pharmingen, and do only frozen sections on 
fresh snap frozen tissues.  You can do either enzyme immunohistochemistry 
or delightful double immunfluorescence staining if you want to see 
colocalization of the antigens.   These antibodies may NOT work on formalin 
fixed paraffin seftions.

Go to BD Pharmingens website for pricing,  quantities, and also product 
data sheets for particulars on these antibodies.

At 02:03 AM 9/28/2006, you wrote:
>I need some recomendation to choose antibody for VCAM-1, ICAM-1 and 
>P-selectin detection on mouse vascular tissue either processed as frozen 
>or paraffin embedded section.  I found so many available products and I 
>have difficulties in choosing. If anyody have experience, please give me
>Histonet mailing list

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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