staining thin sections, GMA vs paraffin,

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From:Gayle Callis <>
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GMA is LESS hydrophobic than methylmethacrylate, with MMA  needing
complete removal for larger molecules to penetrate, that is why low
molecular weight dyes work best with PMMA, and routine stains work better
with GMA. Now for all the blab, blah, blah, blah!   
  I do recall GMA thin sections and a periodic acid procedure, I did it,
years ago, but it was not successful, had to think more in terms of
properties of GMA, and not much there to stain in the first place. 

The periodic acid oxidation was recommended as a way to POSSIBLY improve
nuclear staining, by oxidizing groups on the RNA and DNA, freeing up more
to stain with the hematoxylin, and maybe making the nuclei have darker
staining.  In my experience, that never seemed to work.  Just resorted to
thicker sections, so have more to stain in the first place, better yet,
just did paraffin.  Trying to recall the exact chemistry of oxidizing the
nucleic acids, vicinal OH groups on the sugar, going to CHO groups, but
with overoxidation, creating more COOH groups available to the hematoxylin
stain?  John, comments here.  

In Hrapchak and Sheehans book, 2nd editon, it is recommended that
basophilic properties of nuclei could be restored with 5% periodic acid
overnight, after the following problems, storage in nonbuffered, or
deteriorated NBF, overexposure to decalcifying soltuions, poor washing
after decalcification, or dried/burned tissue.   

I think one needs to read this article, by Gerrits and Horobin, J of
Histotechnology, Dec 1996, 19(4):297-311 titled Glycol methacrylate,
embedding for light microscopy: basic principles and troubleshooting and
understand the penetration of dyes into sections. 

Quickly, large dyes penetrate GMA slowly, penetrating only the components
of tissues that are poorly infiltrated by the plastic.  
Small dyes penetrate GMA easily, and tissue components whether or not
infiltrated by the GMA.  
Intermediate sized dyes penetrate the resin, but stain tissue slowly.
Has to do with molecular weight aka size of dye molecules, and there is
much more to why staining works or not with GMA.

Heat, length of time tend to improve staining with certain procedures, but
read the article, it is an education and at looking at it again, a must for
those working with GMA if you want to understand what is going on.

Paraffin sections, of course have the hydrophobic barrierremoved with
xylene or equivalent, and generally stain freely.

Whew, this was a lot!


Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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