A few years ago I took a workshop at the NSH presented by Neil Hand. He basically provided an account of his experience in working with something like over 100 antibodies used on resin (MMA) embedded bone and possibly other tissues. Maybe you can look his information up on the NSH website and contact him directly?
With regards to an "etching step", there are several posts that describe everything from HCl to formic acid to sodium ethoxide. In fact, I just found this posting from Gayle Callis back in 2004. Hope this helps! Jack [Histonet] Repy on immunostaining with Technovit 8100 / glycol methacrylate Gayle Callis gcallis <@t> montana.edu Thu Aug 12 10:13:55 CDT 2004 GMA is not a very ideal embedding media for immunohistochemistry, you would be far better off with paraffin using this antibody. GMA cannot be removed once polymerized nor sodium ethoxide (generally reserved for electron microscopy resins) etched with much success, you would be better off embedding in methylmethacrylate and remove the plastic entirely. This has been done with great success by Neil Hand (he has publications) using warm xylene, but he used stringent pressure cooker retrieval and worked with human tissue. The problem with GMA is it prevents the immunoglobulins from reaching antigenic sites, as the GMA is hydrophobic plus it can't be removed once polymerized. It will soften in the presence of water. Also, as GMA polymerizes it becomes very hot due to exothermic reaction unless you control this temperature by letting your blocks polymerize on ice in a refrigerator?? GMA with IHC problems have been discussed at length on Histonet many times, go to Histonet archives and search at www.histosearch.org. Personally, I don't think the product "suggestion" is correct, as it only "suggests" but does not say it WILL work. That is probably the reason you don't find it in the literature! Many people have experienced failure of IHC on GMA embedded tissues. I think some have had success with immunofluroescence using immunoglobulins and not a lot of antibodies either. It was a tedious stringent protocol described in a symposium talk. I think you will find in Histonet commentary, that most people attempting IHC/GMA suggest going to another embedding media. Also, CD68 ED1 is a Mouse antiRat, rather than a rat antiMouse (clone FA-11). ED1 and other rat macrophage markers, ED2, ED3, have been discussed on Histonet as FFPE tissues including retrieval for IHC. The staining was very straightforward as was retrieval described and solvents used plus heat of paraffin processing did not damage antigen. ED1, when used in on rat tissue, works well on paraffin sections, although we prefer frozen sections to avoid all aldehyde fixation and no retrieval. When we do frozen sections, the ED1 is diluted out 1:3000 or so, it will not be so dilute for paraffin sections and we detected with secondary to mouse IgG1 isotype (Southern Biotechnology) SA-HRP, and AEC chromogen. Staining pattern is spectacular in a rat spleen, normal positive control. Gayle Callis MT,HT,HTL(ASCP) Research Histopathology Supervisor Veterinary Molecular Biology Montana State University - Bozeman PO Box 173610 Bozeman MT 59717-3610 > To: histonet@lists.utsouthwestern.edu > Date: Fri, 9 Apr 2010 09:11:59 -0400 > From: brusk...@aol.com > Subject: [Histonet] Immuno staining on plastic sections > > > > We are trying to do immuno staining on plastic sections and are not having > much luck. Does anyone have any experience with this? We are embedding in > technovit 8100. It is a device with encapsulated VEGFR cells. I have been > told that we might have to do an etching step. Any help would be greatly > appreciated. > Bruce > > > > _______________________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet