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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

Procedure:

Cut fresh frozen tissue sections at 5-10 um thick and mount on slides.
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.
Let slides air dry for a few minutes.
Place in absolute propylene glycol for 2-5 minutes to avoid carrying water into Oil Red O.
Stain in pre-warmed Oil Red O solution for 8-10 minutes in 60 ºC oven.
Differentiate in 85% propylene glycol solution for 2-5 minutes.
Rinse in 2 changes of distilled water.
Stain in Gill's or Mayer's hamatoxylin for 30 seconds.
Wash thoroughly in running tap water for 3 minutes.
Place slides in distilled water.

• Mount with glycerin jelly or other aqueous mounting medium.
  

Results:

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

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

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Oil Red O Staining Protocol
Prepared byROY ELLISIMVS Division of PathologyThe Queen Elizabeth HospitalWoodville Road, Woodville, South Australia 5011

NovaUltra Special Stain Kits
Principle
The histological mechanism of the staining of lipids is invariably a function of the physical properties of the dye being more soluble in the lipid to be demonstrated than in the vehicular solvent.  The polyazo group of dyes include the oil red series, the Sudan red series, and the Sudan blacks.  All these dyes are interchangeable, and may be substituted in the above method.

Technical Points

1.  Include a positive control
2.  Cryostat sections 8 to 10 microns or formalin fixed smears. Formalin fixation gives good results.
3.  Step 4 - The stain must be freshly prepared from the stock solution, (see below), and kept in a closed container since solvent evaporation will cause stain precipitation.
4.  Step 8 - Obviously, the section must not be taken through clearing solvents prior to mounting, as this will remove the lipid to be demonstrated.

Method

1.
Cut frozen sections at 8 to10mm, air dry the sections to the slides
2.
Fix in formalin, briefly wash with running tap water 1-10 mins
3.
Rinse with 60% isopropanol
4.
Stain with freshly prepared Oil Red O working solution 15 mins
5.
Rinse with 60% isopropanol
6.
Lightly stain nuclei with alum haematoxylin 5 dips
7.
Rinse with distilled water
8.
Mount in aqueous mountant or glycerine jelly.

Results

• lipid......................................red
  
• nuclei....................................blue
  

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Reagent Formulae

1.   Oil red O stock stain
oil red O (CI 26125)       0.5 g
isopropanol                  100.0 ml
Dissolve the dye in the isopropanol, using the very gentle heat of a water bath.  This is the stock stain.
CARE - fire hazard

2.   Oil Red O working solution
For use: Dilute 30 ml of the stock stain with 20 ml of distilled water, allow to stand for 10 mins, and filter into a Coplin jar, and cover immediately.  The stain does not keep, and should be made up fresh from the stock solution each time.

3.   Glycerine Jelly Mounting Medium
      Gelatin (Kitchen grade)    10 g
      Distilled Water                60 ml
      Glycerol                        
70 ml

Phenol                            0.25 g
      Dissolve the gelatine in the distilled water using sufficient heat to melt the gelatine, add the glycerol and phenol. Mix well and transfer to a small capped bottle and refrigerate.