[Histonet] Quantifying lipid in rat liver

[Lee & Peggy Wenk]

Could you fix the tissue in osmium tetroxide, similar to what they do in EM?

- Fix first in 10% neutral buffered formalin to preserve morphology of other
cells, rinse in buffer, then fix in 1% OsO4, rinse again in buffer. That
would fix and stain all the fat black.
- Then process the tissue as usual through alcohols and xylene (or
substitute). Embed in paraffin. 
- Section as usual (might want to put section on charged slides). 5 um would
probably work just fine. Dry slides in oven.

Then, depending upon what you want, either:
- deparaffinize and coverslip with synthetic mounting media
- do an entire H&E on slide. Cytoplasm will look pink-brown, and nuclei will
look blue-brown, but at least you can see other tissue components.

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of David Burk
Sent: Thursday, September 11, 2008 10:26 AM
To: histonet <@t> lists.utsouthwestern.edu; Microscopy <@t> microscopy.com
Subject: [Histonet] Quantifying lipid in rat liver 

Oh esteemed experts who have no peer,

I am in need of advice on how to quickly and easily quantify lipid content
in rat liver.  I believe a traditional approach is to process/embed in
paraffin/section/H&E and then look for the holes left by the lipid droplets.
While this is certainly doable it does require that your samples 'look
nice'.  Unfortunately, many of the sections we have seen here are kind of
banged up and morphology is poor.

I was wondering if you all have another method that you prefer for this type
of analysis.  For example, can I just cryosection and stain with Bodipy or
Oil Red-O?  In those cases I could use the fluorescent nature of the probe
to quantify fat content very quickly on a computer.  Here, damage caused
during sectioning should not create too many problems in quantification as
the stain is specific for lipid.

If we wished to avoid image analysis altogether, could we just drop a rat
liver in an NMR and get content that way?

Thanks so much for all your help!

David Burk

Pennington Biomedical Research Center
Baton Rouge, LA 70808

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