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Oil Red O

For the Detection of Neutral Fats.

Author: Kelsey Goldman, HTL (ASCP)

Last Update: 13 March 2025

Copyright: 2025, HistologyOutlines.com

Cite this Page! -> Goldman, Kelsey. “Oil Red O.” HistologyOutlines.Com, 13 March 2025. Accessed (today).

Purpose

  • Used for the staining of nuetral fats (Triglycerides) in frozen tissues(Disbrey, 1970),(Humanson & Humason, 1979),(Carson, 2001) .
  • Oil Red O staing would allow the viewer to ID fat in an improper place, such as a fat emboli from a crush injury. This would be useful in determining cause of death of a patient as a result of the fat emboli (Carson, 2001).
  • Oil Red O can also be used to ID liposarcomas and degenerated myelin material that has formed droplets (Carson, 2001)

Control Tissues

  • tissue known to contain fat, such as adipose tissue. Most often, internal tissue control is sufficient(Carson, 2001).

Staining Pattern

  • Lipids- Orange Red to Bright Red.
  • Nuclei- Blue

Biochemistry Theory

  • Oil based dyes have a high affinity for lipids. The oil red O is not water soluble (Carson, 2001).
  • The dye is thought to dissolve into the tissue fluid itself. Mixing with but not altering the lipids. The oil Red O does not attach itself to the lipids but rather presents itself in close association (Humanson & Humason, 1979).

Troubleshooting

  • Lipids cannot be stained for in FFPE tissue. While lipids are water insoluble, they are soluble in many solvents like alcohol and acetone. This means that when using normal processing protocols for FFPE tissue the lipids will be forces to seep out of the tissue and therefore are not available for detection (Humanson & Humason, 1979).
  • Gomori argues for a fixation with 10% NBF with 1% Calcium chloride to make the Phospholipids more insoluble (Humanson & Humason, 1979).
  • Do not press on glass when cover slipping (the fat can be displaced and move around the tissue). Rather, soak slide in warm water to displace any air bubbles (Carson, 2001).
  • For Fixation you may use 10% NBF. NO ALCOHOL- lipids are dissolved in alcohol (Carson, 2001).
  • Frozen Slides for this application are typically cut at 10 uM (Carson, 2001).

Additional References

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  • Carson, F. L. (2001). Histotechnology: a self-instructional text (2. ed., [Reprint]). ASCP Press.
  • Disbrey, B. (1970). Histological Laboratory Methods. E.& S. Livingstone.
  • Humanson, G. L., & Humason, G. L. (1979). Animal tissue techniques (4. ed). Freeman.
  • Sumner, A. T. (1969). A Laboratory Manual of Microtechnique and Histology. Blackwell Scientific Publication.

Sample Protocol

    Pearse's Method

    Reagents

    • Oil Red O solution
      • 2.5 g of oil red O
      • 500 mL of 98% isopropanol
    • Oil Red O Working Solution (filtered, allow to stand for 10 minutes after mixing)
      • 24 mL Oil Red O stock solution
      • 16 mL DiH2O

    Method

    1. Cut frozen sections, fix and rinse in tap water, blot off excess.
    2. Place in oil red O working solution for 10 minutes.
    3. Rinse in tap water.
    4. Stain for 1 minute in Harris hematoxylin that contains acetic acid.
    5. Wash in tap water.
    6. Blue in ammonia water.
    7. Wash in tap water.
    8. Coverslip with an aqueous mounting media, seal edges with nail polish.
        9. Adapted from Protocal in Carsons

    Disbrey’s Protocol

    Reagents for the Oil Red O Stock Solution

    • Oil Red O 0.5 g
    • Isopropanol 500 mL

    Protocol to make the Stock Solution

    Place the above reagents into a flask and place the flask into a warm water bath (approx. 56°C). Leave the flask in the bath for 30-60 minutes. Remove and allow the solution to cool to room temperature. This solution should last several months so long as atmospheric water is not allowed access to it.

    Reagents

    • Oil Red O working solution
      • Add 30 mL of your stock solution to 20 mL of deionized water.
      • Mix well. Filter before use.
      • Discard working solution within a few hours.
    • Harris Hematoxylin
    • 0.5% Aqueous hydrochloric acid

    Protocol

    1. Place frozen sections into Oil Red O working solution for 5-10 minutes.
    2. Wash in running tap water for 3-4 minutes.
    3. Stain in Harris hematoxylin for 3-4 minutes.
    4. Wash in running tap water for 5 minutes.
    5. Differentiate in 0.5% Aqueous hydrochloric acid for 5-10 seconds.
    6. Wash in running tap water for 5-10 minutes.
    7. Mount in an aqueous mounting medium.

    Adapted from (Disbrey, 1970)

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