Methods for Lipid Demonstration: Oil Red O
purpose
Oil Red O is used to demonstrate neutral lipids in tissue sections. Because processing alcohols will dissolve lipids from tissue, frozen sections must be used for this method. It can be used to detect such conditions as demyelination, fat emboli, and liposarcomas.
Principle
Lipid demonstration uses physical methods of staining, on the principle of selective solubility. The dye molecule (lysochrome) will leave its solution to absorb into the lipids present in the tissue. The dyes used in these methods must meet the following conditions:
- The dye molecule must be more readily soluble in tissue lipids than it is in the solvent used for the staining solution (isopropanol)
- The dye must not be soluble in water
- The dye must be strongly colored
- The dye molecule must only act in solution
solutions
Oil Red O Stock Solution
- Oil Red O 2.5 g
- Isopropanol, 98% 500 mL
Mix well
Oil Red O Working Solution
Oil red O stock solution 24 mL
Distilled water 16 mL
Mix well, stand for 10 minutes. Filter the solution before use and use within a few hours of preparation.
Mayer Hematoxylin
- Hematoxylin – 1 g
- Distilled water – 1L
- Sodium iodate (oxidizer) – 0.2 g
- Ammonium or potassium aluminum sulfate (mordant) – 50 g
- Citric acid (adjusts pH) – 1 g
- Chloral hydrate (reduces precipitate) – 50 g
Dissolve the hematoxylin in the water, bring to boiling point and boil for 5 min. Remove from heat and add sodium iodate. Allow to oxidize for 10 min. Add and mix completely the remaining reagents in order listed. Cool and filter before use.
Note: We use purchased for student labs
Lithium Carbonate 1%
- Lithium Carbonate 1g
- Distilled Water 100 mL
PROCEDURE
- Prepare fresh working solution of Oil Red O and filter into coplin jar.
- Cut frozen sections of tissue at 10 µm according to established procedure. Fix slides immediately in 10% NBF and wash well in tap water.
- Gently blot excess water from slide, taking care not to disturb tissue section.
- Stain sections in Oil Red O working solution for 10 minutes.
- Wash slides in tap water.
- Stain in Mayer hematoxylin for 2 minutes.
- Wash in tap water.
- Blue sections in lithium carbonate until color change is visible (30 sec – 1 min)
- Wash in tap water.
- Coverslip sections using aqueous mounting medium (Take care not to apply undue pressure on coverslip when chasing out bubbles, as fat droplets may be displaced from tissue)
- To preserve sections for longer time, seal edges of coverslip with clear fingernail polish.
Results
- Neutral lipids: Red
- Nuclei: Blue
control
Most tissue contains some lipid, and so the patient slide can serve as an internal control. If a control slide is needed, a frozen section of various types of fresh tissue can be used.
Reference: Carson, F. L., & Cappellano, C. H. (2015). Histotechnology: A Self-Instructional Text (4th ed.). American Society of Clinical Pathologists Press.