Try using Glycol methacrylate (GMA) with a water gradient processing
instead of alcohol gradient. 

GMA is miscible with water so you can do a different type of tissue
processing or "dehydration".  

Tissue should be fixed in aqueous fixative, rinse briefly to remove
fixative.   Do not use thick samples, 1 mm thickness is ideal for GMA - any
thicker and problems can occur during polymerization. 

1.	Mixture of GMA monomer 20%/water 80% 1 hour
2.	GMA monmer 30%/water 70% 1 hour
3.	GMA monomer 50%/water 50% 1 hour
4.	GMA monomer 70%/ water 30% 1 hour
5.	GMA monomer 80%/water 20% 1 hour
6.	GMA monomer 90%/water 10% 2 changed for 1 hour
7.	GMA catalyzed monomer 1 hour at 4C (refrig) 2 changes or overnight at 4C
	to prevent polymerization at RT.

The tissue will appear very translucent, should not be floating.  Work in
hood, wear double nitrile gloves GMA is not great to get on skin nor
breathe toxic nn dimethylaniline

Do not use vacuum as GMA may unexpectedly polymerize on you and that is
disaster. You might be able cut down on steps 1 and 2.  

8.	Embed in fresh catalyzed monomer with accerlerator.
9. 	Section with a glass knife, tungsten carbide knife OR triangular 
	tungsten carbide knife from DDK (these are great for labs without a 	glass
knife breaker!) 

This method was given to me years ago by Sharon van der Velde - she used
this for brain tissue and/or tissues where alcohols extracted certain
components.  No alcohols are involved at any step since you use a water
gradient. 

 
Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu