Re: Brown fat in plastic/fat stain

From:Sarah Jones

Hi Gayle,
  Do you know of a fat stain that will work in GMA?  Thanks, Sarah

Sarah Jones HT(ASCP)
Dept. of Vet. Anatomy & Public Health
Histology Lab
Texas A&M University
College Station, TX 77843-4458
phone: 979-845-3177
fax:  979-458-3499

>>> Gayle Callis  02/18/03 06:11PM >>>
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 -
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
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
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
this for brain tissue and/or tissues where alcohols extracted certain
components.  No alcohols are involved at any step since you use a

Gayle Callis
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)


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