Re: [Histonet] H&E staining on GMA plastic

From:Gayle Callis

After sectioning, merely dry the sections at 60C.

Gill 3 for 10  min up to 20 min.  We never used Harris hematoxylin, only 
progressive hematoxylins.  Other progressive hematoxylins will work, i.e. 
Richard Allan Hematoxylin 1.  Bone requires more time in hematoxylin and 
less time in eosin/phloxine, this counterstain gives wonderful contrast 
with GMA sections.
Rinse 3 changes distilled water 10 dips each
Blue with Scotts tap water substitute 2 minutes
Rinse 3 changes distilled water 10 dips
eosin/phloxine for 10 minutes, you may find this shorter time even down to 
2 min
Rinse very quickly in 95% ethanol
Rinse very quickly in 100% ethanol
AIR DRY SECTIONS and mount coverslip WITHOUT dipping in xylene.  If there 
any funky wrinkles of plastic close to section, remove carefully with a 
razor blade, sometimes we had curled lips on edges of sections - this way 
you have a really flat coverglass.

Water and solvents (alcohol and xylenes, etc)  soften GMA but do NOT remove 
it,  and will cause really ugly wrinkles and cracked plastic.  We learned 
to coverslip without wetting a slide with solvent.

The joy of GMA, hydrating sections is NOT necessary, you can immerse dry 
sections directly into stains, hence the lovely short protocol.

By the way, I have some gems for GMA staining, a manual from an expert from 
long ago, Sharon van de Velde back in the early 80's and also Nate Brinn - 
he did some very fine work with this plastic, including enzyme 
histochemical staining as did Sharon.

Gayle Callis
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)

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