You wrote: Hi all,
I am going to be exploring some tissue embedding using Glycolmethacrylate (GMA), as I read that it can preserve enzyme function in some cases and potentially can be used for IHC. However, information is a bit scant and can be contradictory at times, to say the least. If anyone has any experience doing any histological or IHC staining on GMA embedded tissue, or knows of anywhere I could get protocols from or any papers that are a bit more recent (I'm mostly finding 90s, early 00s), pleas let me know, I would really appreciate any resources I can find! -- Casey Berridge **************************************************************************** ************************************** GMA references from the 80s and 90s pretty much remain current for GMA. Be sure to do a literature search in J Histochem Cytochem (JHC.org) to see if there are more recent publications. Google Scholar was not turning up much for recent years. We worked with GMA for many years but never for IHC. There are many considerations when using GMA both good and bad with emphasis on the negative side of things from my point of view. We had success when studying some single cell protozoa, including Cryptosporidia in a research setting. There is one publication in the old Stain Technology, now Biotechnic & Histochemistry using GMA for successful enzyme staining. Namba M et al. Improvement in histochemical demonstration of esterase in glycol methacrylate tissue sections by cold temperature embedding in glycol methacrylate. 1983 58(4):207. Several things about GMA. Requires a fume hood in order to work with toxic and carcinogenic chemicals. Glycol methacrylate is sensitizing and several colleagues are so allergic to fumes after working with this plastic over several years, they cant be in the same room where GMA is being worked with. Double gloving is advisable, and wearing safety glasses is a must since the sections are small and can fly into an eye (know of this happening) which is not a good situation. There should be no skin contact with the plastic, nor breathing the n, n, di methylaniline, a carcinogen. Controlling polymerization can be a problem unless you place embedding molds on top if ice, and cooling the embedding mixture with ice water. The polymerization is exothermic, and actually as blocks polymerize gets uncomfortably hot which may be damaging to enzymes and sensitive antigens although the heat can be dispersed. Samples cannot be any thicker than 2 mm, with 1 mm X 1 mm is recommended. This plastic was first used for liver needle biopsies. Polymerization for larger samples is hard to control as is the infiltration by this plastic hence smaller, thinner samples. Sectioning is commonly done with glass knives although tungsten carbide knives work, and I know of one group using disposable blades with a Leica 2255 model microtome. More powerful microtomes i.e. Leica 2650 or equivalent works best. We had a JB-4 microtome with a special block holder to accommodate the metal chucks/block holders sold by Polysciences. The metal block holders were a better heat sink to disperse heat of polymerization. Sections are generally no thicker than 1 to 3 µm, and were wonderful when studying single cell protozoa. We never used GMA for more routine tissue sections although it was popular for bone biopsies in clinical labs over the many years. I personally found it labor intensive, and expensive for our projects although the staining results for H&E, PAS-H and some other special stains very nice. Routine stains can be used, including PAS-H, H&E, Massons trichrome with a modified method, and others. IHC will not work well, even with JB-4 Immunobed. GMA, once polymerized, cannot be removed from the section. Immunobed is probably just a looser matrix than JB-4 and some people have success. GMA plastic is less hydrophobic but still will not allow large immunoglobulins to reach antigenic sites. There has been some success with IHC but in general, GMA is not the ideal embedding media for immunostaining. Neil Hand worked with Poly methylmethacrylate for IHC since the plastic can be completely removed from a thin section, followed by stringent HIER using a pressure cooker. PMMA is another world for processing, sectioning and staining. When doing H&E, the staining protocol is different from paraffin section staining. If you do an extensive, time intensive search on Histonet, there are many discussions about GMA staining both for routine and IHC. You can buy kits, JB-4 and Technovits. The JB-4 discolors over the years to a dark tea/brown color making it more difficult to see the tissue while Technovits remains clear. When you cut sections, you work with one section at a time, not a ribbon. I have a huge file on GMA collected from the early 70s all the way to current years. If you reply to me personally, I can help with references and protocols. In todays world, if wanting IHC on plastic embedded tissue, I would be using Neil Hands recommendations and Poly methylmethacrylate (PMMA) plastic embedding mainly since this plastic is removable. I also have close contact with Neil if you need to visit with him. He is a good plastics guru and has extensive experience with IHC using PMMA. Using this plastic also requires stringent safety precautions for handling the chemicals. Sorry I leaned towards a negative view of GMA. Take care Gayle Callis HTL/HT/MT(ASCP) GCallis Histology Service, LLC Bozeman Montana USA _______________________________________________ Histonet mailing list [email protected] http://lists.utsouthwestern.edu/mailman/listinfo/histonet
