Lipids that exist as fats such as oily and greasy hydrophobic lipids are stained with Oil Red O stain.
This is a fat-soluble dye (lysochrome diazo dye). This stain is mainly used for the staining of triglycerides and lipids on frozen sections and some lipoprotein on paraffin embedded tissues.
Staining should be done in fresh frozen tissues, because fixation in alcohol removes most of the lipids. This stain was discovered by Alexandre Beaudoin in 2004.
Principle
The greater solubility of the dye in lipid substances in tissue sections is the principle of staining procedure.
The Oil Red “O” Stain dissolve in the lipid contents in tissues and give red colour to the lipids in tissues.
Solutions
Saturated “Oil Red” in isopropyl alcohol is the stock dye solution.
60% isopropyl alcohol solution is also necessary.
For the preparation of working stain solution, mix 6 ml of stock solution with 4 ml of distilled water.
Keep the mixture to stand for 10 minutes and filter before use.
- First cut fresh frozen sections of 10-15 µm.
- Rinse the sections in 60% isopropyl alcohol.
- Then stain in “Oil Red O” for about 15 minutes.
- Next differentiate in 60% isopropyl alcohol.
- Then wash in water. After that counter stain with Haematoxylin.
- Then wash with water. Mount with Glycerin jelly.
Observations
Fat in tissue sections stains in orange/ brilliant red colour and nuclei stains in blue colour.
Special consideration
Similar dyes to “Oil Red O” are Sudan III, Sudan IV and Sudan Black B.
When staining with these dyes, positive and negative control slides should be used.
Normally for negative control, paraffin processed tissue such as lung is used.
For the preparation of working “Oil Red O” solution, in some procedures they use 1% Dextrin solution instead of distilled water.
It is necessary to filter the staining solution before use.
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