Re: Brown fat in plastic / fat stain

From:Alex Knisely

Osmium tetroxide post-fixation of formalin- or glutaraldehyde-fixed
material stabilizes fat (against elution by nonpolar solvents) and stains
it pale grey in FFPE materials.  Consult your transmission electron
microscopy colleagues for procedure limitations (OsO4 toxicity, sample
size, etc.).

Alex K

At 21:30 18/02/03 -0600, Sarah Jones wrote:
>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)
Alex Knisely, MD
Consultant Histopathologist
Institute of Liver Studies
King's College Hospital
Denmark Hill
London  SE5 9RS  UK
+44 (0)20 - 7346 - 3125 telefax
+44 (0)20 - 7346 - 4627 office

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