[Histonet] GMA in RE: plastic embedding resins (long)

[Gayle Callis]

Deanna,

You did not say what kind of application you are using  (immunostaining, 
regular staining for light microscopy, Electron microscopy).   This often 
determines the optimal plastic choice before you launch you tissues into a 
resin.

GMA means glycol methacrylate, and is water miscible however cannot be 
removed from a section after the plastic is polymerized.

EMS embed 812, Spurrs, Araldite, EPON are used (in general) for electron 
microscopy ultrathin sections and plastic is not removed for staining, 
unless other than toluidine blue in sodium borate,  pH 9 - 11 is used, or 
methylene blue basic fuchsin.  Unless you are doing EM, these plastics may 
not be ideal.

Are you planning to do immunohistochemical staining?  If so, methyl 
methacrylate can be use instead, but the plastic must be totally removed 
before staining, however, IHC is possible aftert the plastic is gone, 
something that will NOT happen with GMA, it cannot be removed after 
polymerization.

Technovits 7100 and 8100 (EBS) and JB-4  and JB-4 Plus (Polysciences) are 
glycol methacrylate kits, and tissue can be stained with most  water 
soluble stains i.e H&E, PAS, etc, etc  Immunohistochemistry is poor since 
GMA cannot be removed from the tissue once polymerized.  GMA will not allow 
large immunoglobulins to reach antigens in tissue, cannot penetrate the 
plastic.

When choosing GMA,  I would assume either would work well, 8100 or 7100 - I 
know the 7100 sections like a dream!  I am not sure why you say one is 
suited better than another for your area, unless you mean humidity.  There 
are ways to counteract that problem when working with GMA.

Plastic monomers cannot be used on processors and it would take far too 
much monomer to fill reservoirs, plus it is toxic and expensive.   You 
should work in a hood at all times with these plastics, and keep them off 
your skin, they are very sensitizing. ts. Hand processing is generally done 
OR you can use the processor to dehydrate the tissue (2 mm thick, thicker 
may lead to polymerization problems) up through absolute ethanol and then 
do plastic monomer infitrations separately, in a hood and 
refrigerator.   Since tissues are so thin and small, hand processing takes 
very little time with infiltration done overnight in a refrigerator or even 
a few hours without vacuum at RT.  Early polymerization must be avoided, 
hence cold temps.   If you exclude air, the plastic will polymerize BEFORE 
you want it to and there is no going back to recover tissue when this 
happens, been there, had it happen!  Huge tearing of hair!!

Polysciences should have a processing protocol in their technical sheet, 
maybe on their website or you can contact pmarcum <@t> polysciences.com for 
information.   For Technovits -  go to these websites - both are 
processing/handling protocols, very nice and tidy.   EBS sells Technovits

http://www.biol.s.u-tokyo.ac.jp/users/hassei/plotocol/plasti_%20embedding_8100.html 

http://www.ebsstore.com/control/category/~category_id=E1/~pcategory=E

Gamble and Bancrofts Theory and Practice of Histological Technique, second 
edition, 2002  has a whole chapter devoted to using plastics, i.e. GMA.

Be sure to go back in Histonet too, there have been many discussion about 
plastics.  Type in resin names as keywords.

There have also been many excellent articles published over the years in J 
of Histotechnology on glycol methacrylate.  If you have  a medical library 
handy and they have the journal, it is worth some digging out to learn 
about this plastic along with staining methods. etc. J of Histotechnology 
is available on line for at least the issues.

There are special embedding molds from these companies, designed for GMA 
work (Polysciences) and block holders - these are very handy and worth the 
investment, molds are resuable.  You will need extremely sharp knives, we 
prefer glass knives although tungsten carbide. Some have success with 
disposable microtome blades.

Good luck

At 11:46 AM 1/24/2005, you wrote:
>I am trying to learn GMA procedures.  So far I am just reding and geting
>familiar with the process.  I have heard different opinions on different
>embedding medias, ie; EMS Embed 812, Spurrs, and the kit the lab has,
>Technovit 7100.  I was also told by different people that Technovit 8100
>would be better suited for the area I am in.  I would like to have peoples
>comments on the different products out there.  The positive and the negative
>about the different products.  I will be embedding very small, thin biopsies
>of cultured skin substitute.  The processor used is an RMC EMP 5160.
>
>Any and all advice and comments welcome..
>Thanks
>Deanna Snider HT
>Shriners Hospital
>Cincinnati, OH

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)