Oil Red O Staining Protocol


NovaUltra Special Stain Kits


Description: This protocol is for lipid and fat staining on frozen sections. It may not suitable for paraffin embedded tissue sections.


Fixation: 10% formalin (fixation is necessary, damage of unfixed sections has been seen)


Section: Frozen sections at 5-10 um thick


Solutions and Reagents:


0.5% Oil Red O Solution:


      Oil Red O -------------------------- 0.5 g

      Propylene glycol, 100% ----------- 100 ml

      (Propylene glycol is also called "1,2-Propanediol", Sigma Cat# 398039-500ML or Cat# P4347)


      Add a small amount of propylene glycol to the oil red O and mix well, crush larger pieces with stirring bar. Gradually add the remainder of the propylene glycol while stirring. Heat gently until the solution reaches 95 -100 ºC. Do not allow to go over 110 ºC. Overheating will result in high background staining. Filter solution through coarse filter paper (i.e. 25 um filter paper) while still warm. Allow to stand overnight at room temperature. The solution can be stored at room temperature for many years. If precipitate forms in the solution, re-filter.


85% Propylene Glycol Solution:


      Propylene glycol, 100% ----------------- 85 ml

      Distilled water -------------------------- 15 ml


Gill's or Mayer's Hamatoxylin Solution




  1. Cut fresh frozen tissue sections 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.



      Lipids ----------------------------- red

      Nuclei ----------------------------- pale blue


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