Glycerin Antigen Retrieval
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|From:||Scott Whittaker <email@example.com>|
|Date:||Fri, 22 Oct 1999 08:26:11 +0100|
>Date: (No, or invalid, date.)
>From: BB racing <firstname.lastname@example.org>
>Subject: Glycerin Antigen Retrieval
>To: Histonet <email@example.com>
>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.
>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 technique.
>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
Scott D. Whittaker 218 Carr Hall
EM Technician Gainesville, FL 32610
University Of Florida ph 352-392-1184
ICBR EM Core Lab fax 352-846-0251
The home of " Tips & Tricks "
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