Glycerin Antigen Retrieval
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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
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