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Short answer
Oil Red O staining is a histochemical method that colours neutral fat and lipid droplets red in unfixed or lightly fixed frozen (cryostat) tissue sections, fresh smears and touch preps, with nuclei counterstained blue. It needs frozen material because paraffin processing and ethanol or clearing solvents remove most lipids. Published routes differ mainly in solvent (propylene glycol or isopropanol), stain time and how long the stain keeps.
Definition and scope
Oil Red O (Colour Index 26125) is a lipid-soluble dye. The StainsFile protocol based on Chiffelle and Putt describes the method as suitable for demonstrating fats in unfixed or fixed cryostat or frozen sections, whether mounted on slides or free floating. A manufacturer kit describes its purpose as the histological visualization of fat cells and neutral fat, usable only on frozen tissue sections, fresh smears or touch preps, and labels it for research use only, not for diagnostic use.
In all three published routes below, lipid is red and nuclei are blue. The stain is made by dissolving the dye in a solvent (propylene glycol or isopropanol in the routes below) and applying it to the section. Searchers also use the variants “oil red staining” and “red oil o staining”; the latter is a transposition of the same name, and all three refer to this one method.
How it is used in practice
Three openly published routes are summarised below. This page compares them at overview level and does not replace the full protocol of the laboratory or kit you work with.
Oil red o staining protocol variants compared
| Route | Solvent and stain preparation | Stain time | Differentiation | Keeping quality and hazard note |
|---|---|---|---|---|
| Chiffelle and Putt (StainsFile) | 1 g Oil Red O in 100 mL propylene glycol, heated to about 100 C and filtered hot | 10 min in the stain solution | 85% propylene glycol for 2 to 3 min “to clear the background”, then 50% propylene glycol | Stock keeps “a long time (years)” |
| Commercial kit (Abcam ab150678, instructions v4b, April 2026) | Ready Oil Red O solution and propylene glycol supplied; stain heated to 60 C | 6 to 10 min at 60 C, or overnight at room temperature | 85% propylene glycol for 1 min, then distilled water | Kit stored at room temperature; keep away from open flame |
| University histology core facility (frozen sections, v1.0, January 2023) | 0.5 g Oil Red O in 100 mL 100% isopropanol as stock; working solution is 30 mL stock plus 20 mL distilled water, left 10 min and filtered | 15 min | 60% isopropanol rinse | “The stain does not keep and should be made up fresh”; the stock is flagged as a fire hazard |
All three finish with a nuclear counterstain (Mayer’s hemalum or a modified Mayer’s hematoxylin in two of them, Harris hematoxylin in the third) and an aqueous mounting medium. The StainsFile entry specifies cryostat sections cut at 10 um, usually unfixed and mounted on slides; the core facility protocol adds a brief fixation in 4% paraformaldehyde or 10% neutral buffered formalin only when the tissue was not previously fixed.
Related lipid dyes
The StainsFile entry lists alternatives to Oil Red O: Sudan III, Sudan IV and Sudan black B; with other dyes the lipid is shown as orange or black instead of red, and nuclear fast red may be a more appropriate counterstain with Sudan black B. For other stain families on CASRAI, see H&E staining, auramine-rhodamine staining and Ziehl-Neelsen staining.
Where it matters for labs and supply decisions
A checklist for assembling a bench setup, based on the three published routes above:
- Frozen-section capability. The method presumes cryostat sections, so sectioning equipment must be in place first; see what a cryostat is and cryostat vs. microtome.
- Aqueous mounting medium. The kit instructions state that an aqueous mounting medium is required but not supplied; the core facility protocol names glycerine jelly or an aqueous medium.
- Counterstain. The kit supplies a modified Mayer’s hematoxylin; protocols built from separate reagents list their own hemalum or hematoxylin.
- Solvent and fire safety. The isopropanol stock is marked “fire hazard” in the core facility protocol, and the kit says to keep it away from open flame.
- Labelling. The Abcam kit is for research use only, not for diagnostic use.
- Controls. The core facility protocol uses liver as a positive control and paraffin-processed lung as a negative control; the kit names any frozen section containing fat as control tissue.
- Stain freshness. Propylene glycol stock is described as keeping for years, while the isopropanol working solution is made fresh each time, which affects how much is prepared per run.
Common misunderstandings
Can Oil Red O be used on paraffin sections?
Not for most lipids. Paraffin processing removes many lipids, so the StainsFile entry notes that on paraffin sections only solvent-resistant lipids, such as some lipofuscins or residual myelin, can be seen. The core facility protocol uses paraffin-processed lung as a negative control for this reason.
Can slides be dehydrated and cleared before mounting?
No. The core facility protocol states that the section must not be taken through ethanol and clearing solvent before mounting, as this removes the lipids. This is why aqueous mounting is specified.
Does a red result mean a diagnosis?
Not for the Abcam kit: its instructions state it is for research use only, not intended for diagnostic use. A red signal shows where lipid is present in the section.
What does the kit say it detects?
Its instructions describe fat cells and neutral fat, and give any frozen section containing fat as control tissue.
FAQ
Is “oil red staining” the same as Oil Red O staining?
Yes. In laboratory usage the shorter phrase refers to the same dye and method; “red oil o staining” is a word-order variant of the same name.
What is the Oil Red O staining protocol?
There is no single protocol. The propylene glycol route, the kit route and the isopropanol route in the table above differ in solvent, stain time, differentiation and shelf life, but share frozen sections, a nuclear counterstain and aqueous mounting.
What colour is the result?
Lipid appears red and nuclei blue. With the alternative dyes Sudan III, Sudan IV or Sudan black B, StainsFile gives the lipid colour as orange or black.
Does the stain keep?
It depends on the route: the propylene glycol stock is described as keeping for years, while the isopropanol working solution should be made fresh.
Sources
- StainsFile, “Chiffelle and Putt’s Oil Red O for Lipids” (cites Culling, Handbook of Histopathological and Histochemical Techniques, 3rd ed., 1974), https://www.stainsfile.com/protocols/chiffelle-and-putts-oil-red-o-for-lipids/, checked 2026-10-29.
- Abcam, Oil Red O Stain Kit (ab150678) instructions, v4b, updated 21 April 2026, https://content.abcam.com/content/dam/abcam/product/documents/150/ab150678/Oil-Red-O-staining-protocol-book-v4b-ab150678 (website).pdf, checked 2026-10-29.
- University of Saskatchewan histology facility, Oil Red O staining protocol for frozen sections, version 1.0, January 2023, https://healthsciencestest.usask.ca/facility-services/Histology/oil-red-o-staining-protocol-frozen-sections-version-1.0-january-2023.pdf, checked 2026-10-29.
