Oil Red O

Classification: lipid stain

Mechanism of staining: selective solubility

Purpose: stain lipids

Control tissue: any tissue containing fat

Properly Stained Slide

Lipid & lipofuchsin: red


Nuclei: blue






Oil Red O

Primary stain – demonstrates lipids (progressive stain)

Selective solubility. Tissue neutral lipids are more soluble than 60% isopropanol.

Omitted: Lipids will not be demonstrated.

Too short: Lipid staining will be less intense.

Too long: Will not affect staining.

60% Isopropanol

Removes background staining

Organic solvent used to make  a collodial suspension with Oil Red O.

Omitted: Pink background staining. Isopropanol rinse is optional.

Too short: Will not affect staining.

Too long: Will not affect staining.

Harris hematoxylin

Counterstain – stains nuclei and other tissue components



Ionic bonding

Omitted: Nuclei and other tissue elements will not be demonstrated.

Too long: May obscure lipid staining.

Too short: Nuclei and other tissue elements will be obscured.




Special Considerations

Tissues cannot be fixed with alcoholic fixatives that will dissolve the lipids needing to be visualized. Cryostat tissue sections are often used. 

Permount cannot be used to affix the coverslip as it will also dissolve lipids. An aqueous mountant such as glycerol jelly can be used. Glycerol jelly is warmed to 37 degrees celsuis in order to liquify it for application. The edges of a coverslip can be lined with nail polish to further protect the slide. 

Pressure to the coverslip after mounting will cause the lipids to run together, obscuring their origin in the tissue.


Officer B. HIML251 Lecture notes: Congo Red, Toluidine Blue, and Oil Red O, March 11, 2009

Pathology Outlines- Case of Week. 2008. [cited 2009 April 6]. Available from http://pathologyoutlines.com/caseofweek/case200638image5.jpg