Chiffelle and Putt's Oil Red O
for Lipids

This technique may be used for the demonstration of fats in unfixed or fixed, cryostat or frozen sections, whether mounted on slides or free floating. It may also be used for the demonstration of lipids that resist solvent extraction during paraffin processing. Precipitation of the dye onto the glass slide and tissue is not usually seen.

Solutions

Mayer's hemalum.

Solution A
Oil red O	1	g
Propylene glycol	100	mL

Other suitable dyes are:–

Sudan III, Sudan IV, Sudan black B

Add the dye to the propylene glycol, and heat to about 100°C. Mix well until saturated.

Flter while hot using a fast paper. Cool to room temperature, and refilter using a vacuum pump.

This solution keeps for a long time (years).

Tissue sample
Usually unfixed, mounted cryostat sections are used, but formalin fixed, free floating sections are stained satisfactory. Paraffin processed sections may be stained to demonstrate solvent resistant lipids, but see the notes. Sections should be 10 µ thick.

Method

1. Cut and mount cryostat sections on slides.
   Free floating sections may be attached to slides or left free floating.
   Bring paraffin sections to water with xylene and ethanol.
2. Place sections into propylene glycol for 1-2 minutes.
3. Transfer sections into solution A for 10 minutes.
4. Transfer sections to 85% propylene glycol for 2-3 minutes to clear the background.
5. Transfer sections to 50% propylene glycol for 1-2 minutes.
6. Gently agitate in several changes of tap water at room temperature
7. Stain nuclei with solution B for 1 minute.
8. Wash with tap water to blue.
9. Coverslip using an aqueous medium.

Expected results

• Lipids  –  red (orange or black if other dyes were used)
• Nuclei  –  blue

Notes

1. If using sudan black B, nuclear fast red may be a more appropriate counterstain.
2. Although this method can be used with paraffin sections, it will obviously not demonstrate those lipids that are removed by the dehydrating and clearing agents. Only solvent resistant lipids can be seen, such as some lipofuscins or residual myelin.
3. When staining paraffin sections, solution A should be applied for 1 hour or longer, and the handling can be more vigorous.

 

Reference
Culling C.F.A., (1974)
Handbook of histopathological and histochemical techniques Ed. 3
Butterworth, London, UK.

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