Phil, Point taken;  you are right that some lipid is lost. What you would
have to quantitate in my method  is the area of the fat droplets. In my
experience, the fat cells retain enough lipid to show the area quite well
(we used it to demonstrate fat storage diseases). I suppose real
quantitation could be done on Oil-red-O with area and density measurements.
However, since the fat moves around a bit during staining and coverslipping,
it would not be good for morphometric analysis. If all that is needed is
total quantitiation, then non-histology methods would probably  be a better
choice. 

Tim Morken


-----Original Message-----
From: Philip Oshel [mailto:peoshel@wisc.edu] 
Sent: Wednesday, November 10, 2004 9:59 AM
To: Morken, Tim - Labvision
Subject: RE: [Histonet] Oil Red O question


Tim,

I wouldn't trust the quantitation this way. Osmium binds to 
unsaturated  bonds in the lipids, so it doesn't fix saturated lipids 
all that well --  there's still significant loss of lipids during 
dehyration.

Phil

>Andrea, in short, yes, fat can wash away in this stain. If you want to 
>quantitate, I suggest fixing in formalin and then in osmium BEFORE 
>processing to paraffin ( as you would for EM). Then the fat is 
>immoblized and appears dark grey or greenish on the paraffin H&E 
>section. The morphology is good and quanititation is much easier and 
>more reliable.
>
>Tim Morken
>Lab Vision - Neomarkers
>www.labvision.com
>
>Free webhosting for US State Histotechnology Societies: 
>http://www.labvisioncorp.com/demowebsite/index.cfm
>
>-----Original Message-----
>From: Andrea Grantham [mailto:algranth@u.arizona.edu]
>Sent: Wednesday, November 10, 2004 9:25 AM
>To: histonet@lists.utsouthwestern.edu
>Subject: [Histonet] Oil Red O question
>
>
>This may be a crazy question. I'm doing mouse livers for a lab here and 
>they requested Oil Red O. Did the stain - looked great. They are asking 
>if it was possible that some of the fat washed away during the washing 
>steps. The experiment requires that they quantitate the fat found in 
>these sections. Is this a possibility? Is there a better way to 
>quantitate fat? For the record - my washing steps are done under a 
>gentle stream of tap water running into a corner of the stain dish or 
>coplin jar - not a full force gush of water from the faucet directly on 
>the slides. Thanks. Andi Grantham 
>.....................................................................
>: Andrea Grantham, HT(ASCP)     Dept. of Cell Biology & Anatomy     :
>: Sr. Research Specialist       University of Arizona               :
>: (office:  AHSC 4212)          P.O. Box 245044                     :
>: (voice:  520-626-4415)        Tucson, AZ  85724-5044    USA       :
>: (FAX:  520-626-2097)          (email:  algranth@u.arizona.edu)       :
>:...................................................................:
>            http://www.cba.arizona.edu/histology-lab.html
>
>
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-- 
Philip Oshel
Supervisor, BBPIC microscopy facility
Department of Animal Sciences
University of Wisconsin
1675 Observatory Drive
Madison,  WI  53706
voice: (608) 263-4162
fax: (608) 262-5157 (dept. fax)

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