microwave antigen retrieval hints

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From:"Slap, Steven" <SSlap@ebsciences.com>
To:Histonet <histonet@pathology.swmed.edu>
Date:Tue, 07 Sep 1999 15:07:23 -0400
Content-Type:text/plain; charset="iso-8859-1"

Dear HistoNetters,

Kerry Beebe offered some interesting suggestions for microwave antigen
retrieval.  Let me just make a few comments.  Kerry mentions leaving
blank spaces between slides to correct for uneven heating within a
container (evidenced by slides with inconsistent results).  This is an
artifact of insufficient microwave penetration, and can be fixed by
either of two remedies:  using a smaller container (as Kerry did with
his multiple Coplin jars) or agitating the fluid in the container
(simply taking the container out and shaking it works, although it's not
the most elegant solution).  Kerry's problem with multiple Coplin jars
giving inconsistent results is an artifact of a different problem-
relative hot and cold spots within the cavity-  that can, ironically,
also be fixed using a single, larger, container with agitation. 

Best regards,
Steven Slap
Energy Beam Sciences, Inc.
The Laboratory Microwave Company
Adding Brilliance to Your Vision
http://www.ebsciences.com <http://www.ebsciences.com> 

	-----Original Message-----
	From:	BB racing [SMTP:bbracing@silk.net]
	Sent:	None
	To:	Histonet
	Subject:	Glycerin Antigen Retrieval

	This is a new technique for Antigen Retrieval, and is for all of
you out there, with open minds, that like to try new ideas.
	Current antigen retrival techniques revolve around the use of
water,  raised to approximately  120C,  by various means such as
autoclaving, pressure cooking, or microwaving.  There are several
problems associate with this technique that I thought need to be
addressed.  First, is the partial distruction or even complete loss of
the sections from the slides due to the formation of steam bubbles
between the glass and the tissue.  This can causes parts of the sections
to be literally blown off the slide as these bubbles form.  Secondly,
very hot water or steam, causes the collagen, and other connective
tissues to swell during the recovery  process, and again leads to
section distortion, and a general loss of good morphology.  This is
particularly troublesome when the tissues are not as well fixed or
processed as we would always have liked. Thirdly, steam heating is
inherently dangerous and can cause sudden and unexpected burns.
	So, why not use a different solution for antigen retrieval?  One
that has a very high boiling point, does not give off noxious fumes when
heated, does not transfer it heat quickly when spilled, is not toxic, is
stable, reusable, easy to come by,  and most important, will not damage
the tissues sections when heated.
	Glycerin, a polar molecule, with a  290C boiling point came to
mind.  Pure glycerin however failed to work, and it was only when a
small amount of water, (10%) was added that excellent results were
obtained.  This reinforced the concept, that water in some form is
required for antigen retrieval to work, either to assist in the cleaving
off of the formaldehyde molecule from the tissue proteins, or to break
up the methylene bridges, or to rehydrate the tissue proteins we are
interested in detecting. (Dry heat verses wet heat discussion of a few
weeks ago by Mark Lewis and others)  It is interesting to note, that
only a slight, but still noticeable improvement in the retieval results
was obtained when a commercial antigen retrieval solution was used in
the glycerin, so I think that the role that various salts play in
antigen retrieval is still open to much debate.

	The following is the method we are now using for our 1D5's
Estrogen receptor antigen retrieval, as this is where the most dramatic
improvement in antigen recovery,  staining and tissue morphology has
been expirenced.  Other antigens are also nicely recovered but the
results are less dramatic, except for the improved tissue morphology.

	Retrieval solution.
	Glycerin -----  90 mls
	Dako's 10x antigen retrieval solution ---- 10ml

	1.  Place 100 mls of solution and slides in a 100ml plastic
coplin jar.
	2.  Microwave on High for 1.0 min
	3.  Microwave on Low for 10.0 min
	4.  Let cool to room temp for 20.0 min
	5.  Rinse in saline and continue with your usual immuno

	1. My microwave is a 600 watt unit, and it takes 1.0 min for it
to heat the Glycerin solution to 125C. Other microwaves may vary in the
time it takes for the solution to reach this temperature and this is the
time that should be used in the first step.
	2. At low power my microwave maintains the solution at 125-135
C.  Other microwaves may vary in power and the levels and times may have
to be adjusted to compensate for this.
	3.  Due to the nature of microwave radiation, I have to leave
one empty slide space between our slides in order to avoid "cold spots"
in the heating solution which will cause variations in the retrieval of
the antigen.  I have had the same problem with my autoclave, and
corrected it by leaving a space between the slides.  Other microwaves
may or may not have this problem as may other autoclaves.
	4.  When I have more slides than can fit into one coplin jar, I
microwave each jar separately for one minute, then place them all into
the microwave for the second heating, and cooling period.  For some
reason, we get wild temperature variations between the coplin jars when
trying to heat more than one, at a time, on the high setting.

	In conclusion, give it a try, as its easy and simple to do, and
in my hands has given me the best, most consistent, antigen retrieval,
espesially for estrogen receptors, of any technique I have ever used.

	As a working supervisor, who is expected to carry a full bench
every day, I get very little time to do R&D, but I will bet, that there
is probably an even better non aqueous antigen retrieval solution out
there somewhere, that will not affect tissue morphology in any way.
Someone with more time on there hands than I have, just needs to think
of what it might be.

	Kerry Beebe A.R.T.
	Kelowna General Hospital
	Kelowna B.C. Canada


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