Sudan III Stain of Cells in Culture

From:Katherine Brittingham <>


I am doctoral student working in an Immunology lab.  I have no past 
experience in histological stains and I am searching for protocols for 
staining macrophages growing adhered to tissue culture plates.  The goal is 
to induce foam cell formation with oxidized LDL, then stain lipids with 
Sudan III Red (Sigma Chemicals), nucleus with hematoxylin, counterstain with 
leukocyte acid phosphatase (Sigma kit), and then take some pretty pictures 
using a light microscope for publication. I would greatly appreciate any 
advice on the following:

While these stains are bound to be cook-book, in all of my searches, I have 
only turned up protocols for stains on embedded tissue sections (even my 
Acid Phosphatase, Leukocyte Sigma kit instructions), and nothing for cells 
growing in culture (RPMI 1640), so I'm trying to find a protocol that will 
work on cells adherent in a tissue plate.

There appears to be a bunch of different hematoxylin stain protocols.  How 
do I decide which is the best one under these conditions? Which would be the 
best protocol in your opinion?

In addition, I was originally wanting to stain the intracellular lipids with 
Oil Red O, but got mislead by the Sigma online catalog which made it sound 
like Sudan III was another name for Oil Red O, so I accidentally bought 
Sudan III.  I didn't realize the mistake I made until I was reading about 
the different lysochromes on the Stainsfile website.  On that website, the 
authors stated that Sudan III was a more orangy color.  Is it really going 
to make much of a difference in this case if I stain with Sudan III instead 
of Oil Red O, or should I  just bite the bullet and buy Oil Red O (from 
which company?)?  In addition, I'm looking for appropriate protocols.  
Journal articles are really basic in details of the stains.  Usually they 
just say that they did and Oil Red O and leave it at that, which doesn't 
help me.

I would be really greatful for any help or info that would steer me in the 
right direction.  Please let me thank you in advance.

By the way, the histonet is really a great idea!  I wish immunologists had a 
forum like this where there is a free-flow of helpful info.

Katherine Brittingham
University of Louisville
Louisville, KY
lab: (502) 852-6979
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