[Histonet] Repy on immunostaining with Technovit 8100 / glycol methacrylate

[Gayle Callis]

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

At 10:04 PM 8/11/2004, you wrote:

>Hi Histonetters.
>
>I'm trying to immunostain some sections of rat tissue embedded in 
>Technovit 8100 (a glycol methacrylate) with an ED1 antibody (Serotec rat 
>anti mouse), and have had no success to this point. Have tried various 
>antibody concentrations and antigen retrievals including pronase, trypsin 
>(which we use successfully for immunostaining with the same Ab on 
>paraffin-embedded tissues) and high temperature citric acid buffer 
>retrieval. Our samples are paraformaldehyde fixed overnight, and we use a 
>labelled streptavidin-biotin system. The technical information that comes 
>with the product suggests it is suitable for immunohistochemistry (and 
>this is why we purchased it, as well as its water miscibility and lack of 
>requirement for heat or solvents during processing / embedding) but I been 
>unable to find any reference to its use in this way in the scientific 
>literature, or on the net in general, so far. Can anybody give me any 
>suggestions at all as what I might try, or how I might be able to find 
>useful information on immunos with glycol methacrylate in general or 
>Technovit 8100 in particular? I did see recently an archival Histonet 
>posting on "etching" of glycol methacrylate sections using a cured (ie 
>aged) NaOH solution - does anybody have an opinion on this?
>
>Many thanks,
>
>Jason Palmer
>
>Bernard O'Brien Institute of Microsurgery
>42 Fitzroy St, Fitzroy Victoria 3065
>Australia
>tel +61 3 9288 4018
>fax +61 3 9416 0926
>email: palmerj <@t> svhm.org.au
>
>
>
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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)