Re: (Fwd) K. Beebe's Glycerin Antigen Retrieval
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|From:||"J. A. Kiernan" <firstname.lastname@example.org> (by way of histonet)|
It is worth mentioning also that Kerry Beebe's retrieval
procedure is available in print. The article is:
Beebe, K. 1999. Glycerin antigen retrieval.
Microscopy Today 99-9, 30-31. (That's the September issue.)
John Kiernan, London, Canada.
On Mon, 20 Dec 1999, Greg Dobbin wrote:
> ------- Forwarded message follows -------
> >From: BB racing <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.
> >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 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
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