Re: [Histonet] oil red o

From:Rene J Buesa

Hi Sally:
  The following is the Oil red O (ORO) stain I used for more than 20 years, with consistent results always.
  1- Stock solution: ORO dye---1.0 g + isopropyl alcohol ---500 mL. Let stand for at least 24 hours to dissolve. This solution is stable if capped well.
  2- Working solution: ORO stock solution---24 mL + distilled water ---16 mL; mix, cover and let it stand for 10 minutes. Filter through 2 thicknesses of paper because the dye will precipitate on exposure to air (be patient, it will take sometime to filter).
  The staining procedure:
  1- cut the frozen sections.
  2- place the slide with the frozen sections in a Coplin jar with 5 mL of "pure" formaldehyde in a way that the sections are not in contact with the 36% formalin (5 mL will be enought). Cover the jar and place it in a water bath at 45-50 Celsius for 10-15 minutes. The idea is that the sections are fixed by the formalin fumes, not by the liquid itself. After placing the Coplin jar in the water bath, start preparing the ORO working solution.
  3- Take the slides and wash them with distilled water to eliminate the OCT used to embed the tissue for the frozen sections.
  4- Stain the section with the hematoxylin ("Harris" type) at this moment for 30 secs.
  If you try to stain the nuclei after the ORO it will be weaken, so nuclear staining has to be done before the fat stain.
  5- Place the slides in running tap water for 5 minute.
  6- Wash slides with distilled water.
  7- Place the slides in the working ORO solution for 10-15 minutes.
  8- Wash the slides with distilled water, quickly, and cover with a water soluble medium that will harden after a few hours. Do NOT press the coverslipp trying to eliminate bubbles, rather use a "generous" amount of the mounting medium to assure there are no bubbles, and clean around the coverslip afterwards.
  There are many water soluble media in the market but I used one prepared "in house":
  A-Gelatin (same as used for the water bath)---40 g + distilled water--210 mL. Let the gelatin soak in water for 2 hours.
  B- Glycerine ---250 mL + phenol in crystals ---1.0 g.
  Mix A and B and heat gently until all the gelatin is dissolved and the solution is clear. Store in refrigerator.
  This protocol served me well for years, I hope it will be useful for you also!
  Rene J.

Sally Prouty  wrote:
  I am trying to locate a protocol for a consistant Oil red O stain. I don't 
know if it is in coverslipping where the stain is moving off the fat cells 
or in my differentiation step. I am staining on frozen skeletal muscle 
sections. Any suggestions would be helpful. Thank you,


Sally J. Prouty
HHMI/University of Iowa
Kevin Campbell Laboratory
400 EMRB
Iowa City, Iowa 52246

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