[Histonet] RE: [IHCRG] HIER

[Patsy Ruegg]

I have used many different hier methods over the years and have special
considerations because of my altitude.  We thought that the pressure cooker
would solve all the problems relating to altitude because it is pressurized
but it did not.  There must be some boiling going on sometime because I
often got damaged tissue with the PC.  My heat source of choice now includes
either a waterbath or steamer, and I use the steamer most often because
water boils rapidly above about 92c for me, so I have to keep the waterbath
below that.  My husband drilled a hole in the lid of the steamer where I
insert a thermometer.  I use a digital cooking therm., it has a feature that
you can set to alarm when it reaches a programmed temp, I put tubs or coplin
jars with the buffers in the steamer to warm up while I deparaffinize the
slides.  I put the temp probe in the solution in the steamer and set it to
alarm at 90c.  after I put the slides in the buffer in the steamer, I set
the alarm again to go off when it again reaches 90c, (it can take up to
10-15 min. to get back to temp after adding the slides) after it reaches 90c
with the slides in there I start timing, I steam my slides for 15-20 min
after they reach 90+c., they will go up only to about 95c in my lab because
of the altitude I assume.  When they are done I take them out of the steamer
and let them cool on the counter for 20 min or so, then rinse in water and
then place in buffer before doing IHC. 

Patsy

 

Patsy Ruegg, HT(ASCP)QIHC

IHCtech

12635 Montview Blvd. #215

Aurora, CO 80045

720-859-4060

fax 720-859-4110

pruegg <@t> ihctech.net

www.ihctech.net <http://www.ihctech.net/> 

www.ihcrg.org    

 

 

  _____  

From: ihcrg <@t> googlegroups.com [mailto:ihcrg <@t> googlegroups.com] On Behalf Of
Perry, Margaret
Sent: Wednesday, October 22, 2008 10:28 AM
To: ihcrg <@t> googlegroups.com; histonet <@t> lists.utsouthwestern.edu
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 Dakota State University

Box 2175 North Campus Drive

Brookings SD 57007

 


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