[Histonet] Best Ever Oil Red O Protocol

Donna Emge djemge <@t> aol.com
Fri Jan 28 15:06:07 CST 2011


Ada Feldman sent me an article on Oil Red O staining and I wrote the
following protocol for my lab based on the article. It works exceptionally
well, frozen adrenal controls have brightly stained positive cortex and
clear negative medulla. Best of all it has no precipitate like all the other
methods. It also birefringent under polarizing filters. This is the best Oil
Red O protocol anywhere! 


Our lab has tried several water based mounting mediums and the warm glycerin
jelly works best. With glycerin jelly the stain and hematoxylin fades very
little, even after a week. Also the cover glass doesn’t slide around because
the jelly firms up when it cools.




From:  Ada T. Feldman and Richard W. Dapson (1974) Medical Laboratory
RED O, Department of Biology, University of Michigan-Flint, Flint, Michigan
48503, U.S.A.


Triethyl Phosphate (Spectrum T2256, 500ML, CAS 78-40-0) VWR cat. #

Oil Red O (Alfa Aesar CAS# 1320-06-5, 25G, C.I. 26125) VWR cat # AAA12989-14

Glycerin Jelly Mounting Medium (Electron Microscopy Sciences, Cat. #
17998-10, 100 ml) VWR cat# 17998-10

VWR Harris Hematoxylin (VWR Premium Stains, 95057-858) VWR cat # 95057-858 

Lithium Carbonate (TCI America, 500 G) VWR cat TCL0224-500G


Disposable Transfer pipettes VWR cat# 16001-180

VWR Superfrost Plus Microscope slides (VWR 25X75MM PK72) VWR cat# 48311-703

VWR® Micro Cover Glasses, Rectangular, No. 11/2   VWR cat# 48393-194




60% Triethyl Phosphate

600 ml Trithyl Phosphate

400 ml Distilled Water


0.5% Oil Red O in 60% Triethyl Phosphate

300 ml 60% Triethyl phosphate

1.5 gm Oil Red O


Harris Hematoxylin


Saturated Lithium Carbonate 

18 gm lithium carbonate

1500 ml Distilled Water



1.       Pre-warm Glycerin Jelly in 60°C waterbath 30mins prior to staining.

2.       Bring frozen sections to room temperature. 

3.       60% Triethyl Phosphate – a few dips.

4.       Stain sections in 0.5% Oil Red O, 15 to 20 minutes.

5.       Rinse in  water 2 minutes.

6.       Counterstain in filtered Harris Hematoxylin, 2 minutes.

7.       Blue in saturated Lithium Carbonate solution, 10 seconds

8.       Rinse in water 5 minutes and hold in water.

9.       Mount with warm Glycerin Jelly using a clean transfer pipette. Take
care to avoid contaminating the pipette and Glycerin Jelly.


 Fat – Pink, Red, Bright Orange

Nuclei - Blue



Donna J. Emge, ASCP-HT

Mouse Histology and Phenotyping Laboratory Manager

Northwestern University

Olson Pavilion 8-333

710 North Fairbanks Court

Chicago, IL  60611

d-emge <@t> northwestern.edu

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