Oil Red O Staining Protocol


Prepared by
IMVS Division of Pathology
The Queen Elizabeth Hospital
Woodville Road, Woodville, South Australia 5011

NovaUltra Special Stain Kits


The histological mechanism of the staining of lipids is invariably a function of the physical properties of the dye being more soluble in the lipid to be demonstrated than in the vehicular solvent.  The polyazo group of dyes include the oil red series, the Sudan red series, and the Sudan blacks.  All these dyes are interchangeable, and may be substituted in the above method.

Technical Points


1.  Include a positive control

2.  Cryostat sections 8 to 10 microns or formalin fixed smears. Formalin fixation gives good results.

3.  Step 4 - The stain must be freshly prepared from the stock solution, (see below), and kept in a closed container since solvent evaporation will cause stain precipitation.

4.  Step 8 - Obviously, the section must not be taken through clearing solvents prior to mounting, as this will remove the lipid to be demonstrated.




1.      Cut frozen sections at 8 to10mm, air dry the sections to the slides

2.      Fix in formalin, briefly wash with running tap water 1-10 mins

3.      Rinse with 60% isopropanol

4.      Stain with freshly prepared Oil Red O working solution 15 mins

5.      Rinse with 60% isopropanol

6.      Lightly stain nuclei with alum haematoxylin 5 dips

7.      Rinse with distilled water

8.      Mount in aqueous mountant or glycerine jelly.



  • lipid......................................red

  • nuclei....................................blue

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Reagent Formulae


1.   Oil red O stock stain
oil red O (CI 26125)       0.5 g
isopropanol                  100.0 ml
Dissolve the dye in the isopropanol, using the very gentle heat of a water bath.  This is the stock stain.
CARE - fire hazard


2.   Oil Red O working solution
For use: Dilute 30 ml of the stock stain with 20 ml of distilled water, allow to stand for 10 mins, and filter into a Coplin jar, and cover immediately.  The stain does not keep, and should be made up fresh from the stock solution each time.


3.   Glycerine Jelly Mounting Medium

      Gelatin (Kitchen grade)    10 g
      Distilled Water                60 ml

      Glycerol                          70 ml
Phenol                            0.25 g

      Dissolve the gelatine in the distilled water using sufficient heat to melt the gelatine, add the glycerol and phenol. Mix well and transfer to a small capped bottle and refrigerate.





Lillie RD and Ashburn LL,(1943), Supersaturated solutions of fat stains in dilute isopropanol for demonstration of acute fatty degeneration not shown by Herxheimer’s technique. Archs.Path.,36,432