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Special Stains - Oil Red O

  • Lipid Staining technique - Oil Red O
  • Oil Red O Technique

This technique is performed on frozen tissue sections.  Exposure of tissue containing lipid to solvents such as alcohol leads to loss of lipids.  After cutting the tissue onto a charged slide, to prevent washing off, it is left to dry before the technique is carried out.  Oil red O stains up the fats red by using an alcoholic stain solution as this is more soluble in the lipids than in the alcohol leading to stain uptake by fats. This is called selective solubility.

 

    Oil Red O procedure

  1. Cut fresh frozen tissue sections in cryotome at 5-10 um thick and mount on slides.
  2. Air dry slides for 30-60 minutes at room temperature and then fix in ice cold 10% formalin for 5-10 minutes. Air dry again for another 30-60 minutes or rinse immediately in 3 changes of distilled water.
  3. Let slides air dry for a few minutes.
  4. Place in absolute propylene glycol for 2-5 minutes to avoid carrying water into Oil Red O.
  5. Stain in pre-warmed Oil Red O solution for 8-10 minutes in 60 ºC oven.
  6. Differentiate in 85% propylene glycol solution for 2-5 minutes.
  7. Rinse in 2 changes of distilled water.
  8. Stain in Gill's or Mayer's hamatoxylin for 30 seconds.
  9. Wash thoroughly in running tap water for 3 minutes.
  10. Place slides in distilled water.
  11. Mount with glycerin jelly or other aqueous mounting medium

Results

Lipids

Red

Nuclei

Pale Blue

Photo – x10 Oil red on frozen unfixed tissue