4/16/96

Fellow microscopists,
A colleague needs to embed nasal cavities from mice for immunocytochemistry. His antigens are fragile and do not whistand the standard embedding procedure with paraffin. Cryosectioning works but would preferably be avoided because of the bone tissue and decalcification has also proven detrimental to the antigenicity of his preparations.

I suggested to try glycol metacrylate but I have no hands on experience with this material. In addition, there is a variety of commercial kits available, e.g. Polysciences' Immunobed or JB4, but the differences are not exactly obvious to me.

Can anyone recommend a particular preparation of this medium? What are the pros and cons? Is there some good alternative?

Any info and/or references would be welcomed. Thanks a bunch in advance. Regards,

Michel Deschuyteneer, Ph.D.
deschuyt@sbbio.be

Michel,
I have used JB-4 for regular histology of the mouse skull base and had good luck with it for toluidine blue stain only. I use 15-20 ml breakaway molds, 0.7 gm catalyst to 100 ml of soln. A, and 1.0 ml of solution B added to the embedding mixture of 100ml soln. A/0.7 gm catalyst. I put the molds into icewater to start the embedding procedure. I then overfill the molds, place the specimens into the soln., cover the surface with parafilm-making sure to force all the air bubbles out from under the parafilm, seal the edges of the parafilm to the molds with a hot spatula, and place the tray of ice water/molds into a refrigerator overnite. If heat is a problem for your or your friend's immunohistochemisty, the operation stated above must be done quickly, as the polymerization of JB-4 is exothermic. The next day, I remove the bocks from the refrig. and let them stand overnite at room tempurature before I remove the molds. You might need to let the blocks stand for a few more days before you attempt to cut them also. I cut mine on a ralf knife made from a glass microslide ( the cheaper the better) and I cut them on a standard rotary microtome with a razorblade holder to hold the ralf knives. As I stated above, I have not been able to get good staining with any stain other than toluidine blue. If anyone out there has suggestions on how to get any other stain to work with this material I'd like to know.

Karen Pawlowski
: kna101@utdallas.edu

:I haven't tried JB4 or glycol methacrylate for immuno work, but have used butyl methyl methacrylate - it works well for plant tissue - for localising microtubules, actin and callose at least. You need to add 5-10 mM DTT to the fixative and to the embedding resin, seems to preserve the antigenicity. The reference is Baskin et al. 1992 Planta 187: 405-413 (though the procedure we use is modified from this), in which earlier work is cited also. Best results are with UV polymerisation in the cold, though some people get away with room temperature polymerisation.

rgwhite@vaxc.cc.monash.edu.au
(Rosemary White)

These kinds of questions are addressed in the following two publications:

Stirling, John W., Histochemical Journal 24, 190-206 (1992)

Gerrits, Peter O., Eppinger, Bernhard, van Good, Harry, and Horobin, Richard W., Cells & Materials, 1, No.3, 189-198) 1991

There can be some major differences in different GMA or HMPA based kits coming from different sources. The first of the two above papers gives specific mention about "low acid" GMA. The exact acid level can depend on the purification process being used, the quality of the packaging, and of course age of the product and the conditions of storage. The acid level can make a big difference in the quality of the final results. For LM, the acid component shows up as much higher levels of background staining.

Standing in the SPI Supplies exhibit booth at trade shows might not be the most perfect of scientific methods to draw conclusions, but the impression I get is that because GMA has a viscosity like water, when it comes to "bone tissue", it infiltrates quite nicely. Some have told me "better than anything else". Another advantage of GMA is that absolutely no alcohol dehydration is needed, since the monomer acts as its own dehydrator. This is the presumed explanation for there being a generally greater retention of antigenicity than with other commonly used resins. The biggest perceived negatives associated with the use of GMA seem to be the longer than normal learning curve frequently reported in terms of learning how to work with it the first time. Toxicity is often times cited as a negative, however, relative to most of the other acrylates used in microscopy, it surely is not any worse, and some have told me it does not effect them as much. Cost is also a factor since the GMA and HPMA kits are certainly more expensive than some alternative resin systems.

Disclaimer: SPI Supplies is the original commercial supplier of low acid GMA and HPMA kits for both light and electron microscopy and we would love to see more people enjoying the benefits of low acid GMA and low acid HPMA embedding.

Charles A. Garber, Ph. D
GVKM07A@PRODIGY.COM

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