[Histonet] Quantifying lipid in rat liver

[Tony Henwood]

You could try this:

Linoleic Acid Method for the demonstration of Lipids in Paraffin
Sections

Tracy & Walia (2002) have described an ingenious method to fix lipids
for staining fat embolism in paraffin sections:

(Tracy, R.E., Walia, P., (2002) "A Method to fix lipids for staining fat
embolism in paraffin sections" Histopath 41:75-79)

Solutions:

1.	Saturated solution of linoleic acid in 70% ethylene glycol
To 500ml of 70% ethylene glycol, add 5g linoleic acid and 2g lecithin,
mix for one hour and stand for several hours. Draw off the lower phase
in a separatory funnel

2.	2% chromic acid

3.	70 % ethanol

4.	5% Sodium bicarbonate

Method:
1.	Place 2mm thick sections in solution 1 for 3 days at 56oC
2.	Rinse for 8 hours in 70% ethanol
3.	Rinse in water for 8 hours
4.	Immerse in 2% chromic acid for 24 hours at 4oC.
5.	Rinse in water for 24 hours
6.	Place in 5% sodium bicarbonate for 24 hours
7.	Rinse in water 8 hours
8.	Process from 70% ethanol via ethanol and xylene to wax.
9.	Cut paraffin sections and stain with the usual fat stains (eg
oil red O) and mount in an aqueous mountant.


Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145 




-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of David
Burk
Sent: Friday, 12 September 2008 12:26 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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