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Oil Red O
LIPID
In Histotechnology, the word lipid refers to all fat and fat like,
or fat containing substances.
Examples: Triglycerides, fatty acids, lipoproteins and glycolipids.
Classifications:
1. Simple Lipids (Neutral fats) –Esters of fatty acids with alcohols and usually found in
the body as energy stores in adipose tissue.
2. Compound Lipids –consists of fatty acid, alcohol and one or more other groups such
as phosphorus or nitrogen.
a. Phospholipids –important component of cellular membranes particularly found in
mitochondria and nervous tissue elements.
b. Glycolipids -composed of fatty acids and hexoses, possessing characteristics of
both lipids and carbohydrates
3. Derived Lipids –Fatty acids that are derived from hydrolysis of
simple and compound lipids
Adipose Tissue (Fat)
1. White Adipose Tisssue (WAT) –Composed of unilocular lipid-filled adipocytes that specialize in lipid
storage.
2. Brown Adipose Tissue (BAT) –Composed of multilocular adipocytes that specialize in lipid burning.
Fat cells
Appear as “signet rings” on H&E stain.
Lipid Bodies (Lipid droplets or adiposomes)
Distributed in cytoplasm as roughly spherical organelles lacking a delimiting classical bilayer
membrane but surrounded by an outer monolayer of phospholipids, which at least in some cells
may have a unique fatty acids composition.
Neutral lipids (triacyglycerols or cholesteryl esters).
Can be destroyed by drying of fixation and staining with alcohol-based reagents.
Lipids
Difficult to demonstrate histologically
Best demonstrated on cryostat sections of fresh unfixed tissues.
Lipids bound to other materials.
Lipochrome (lipofuscin) pigments
• Breakdown products from oxidation of lipids and lipoproteins.
• Commonly found in heart, liver, CNS & Adrenal cortex.
• PAS positive & variably acid fast
• Stain with Ziehl-Neelsen
• Sudan black negative & Sudan red positive
• Can be demonstrated by Schmorl’s method Lipochrome (lipofuscin) appearing brown pigments. H&E stain of liver
• May exhibit strong orange auto fluorescence in formalin-fixed, unstained paraffin sections.
o Lipids present in fat embolism, fatty liver & atheroma may be fixed for staining in paraffin sections by exposing
the sections to an emulsion of linoleic & lecithin in 70% ethylene glycol at 56 °C for 3 days.
o Theses tissue are then treated with 2% chromic acid at 4 °C for 24 hr in 5% sodium bicarbonate, with
appropriate rising between solutions.
o Paraffin sections of these tissues then stained with a lipid-soluble dye.
Potassium dichromate or Osmic acid
• Treatment for Phospholipids and Neutral fats during routine dehydration and
Embedding.
• Only agents that truly fix lipids.
• Renders phospholipids as non-extractable by alcohol, toluene, xylene.
• However, they greatly alter the chemical reactivity of lipids, which can
adversely affect staining.
Formol-calcium
• Fixative of choice for lipid histochemistry
• Prepared by addition of 2% calcium acetate to 10% formalin.
Polyethylene glycols (Carbowaxes)
• Sometimes used instead of usual dehydration and embedding process
to preserve lipids.
• Not widely utilized
• In most centers, neutral fats are still best demonstrated in frozen
Sections of unfixed tissue.
Formol-calcium
• Also suitable for cryostat sections
• Should be mounted on chrome-gelatin coated slides
(fixation causes the endogenous tissue proteins to lose their adhesive properties)
FAT STAINS AND SUDAN DYES
Sundanophilia
- Property of tissue to be stained with fat or oil-soluble dyes.
The staining is based on the greater physical solubility of the dye in lipid substances than the usual aqueous-
alcoholic or acetone-alcoholic medium in which they are dissolved.
Staining with these dyes is regarded as specific for lipids, especially for simple lipids.
Oil soluble dyes are divided into the main groups:
1. Basic Aryl amines with very low water solubility:
o Sudan Black B – most sensitive lipid stain known
o Sudan Red VII B
1. B-Naphthols such as the original diazo dyes
o Sudan III (C.I. No. 26100)
o Sudan IV (Scharlach B) C.I. No. 26105 – staining fats with a more brilliant or deeper red color than Sudan III
shich stains lipids orange-red.
Sudan dyes
Group of lipid soluble solvent dyes often called lysochromes.
Sudan III - first of these dyes to be introduced in 1896, followed by Sudan IV (Scharlach R) in 1901.
Predominantly used for staining tryglycerides in animal tissues (frozen sections).
With the use of other solvents, may also be used to stain some protein bound lipids in paraffin sections.
Sudan Black B - most sensitive and versatile.
Introduced in 1935
Stains phospholipids as well as neutral fats
However, it does not stain crystalline cholesterol, and free fatty acids (tend to be dissolved in the alcoholic dye bath).
These drawbacks can be overcome by pre-treating the tissue with bromine to make the unsaturated lipids insoluble in
organic solvents.
For frozen sections, cut tissues about 15 micra thick are usually stained with Scharlach R or with Oil Red O, which stains
neutral fats and lipofuschin well.
Sudan dyes
Oil Red O – used to demonstrate the presence of fat or lipids in fresh, frozen
tissue sections.
Fat-soluble diazo dye
Classified as one of the Sudan dyes which have been in use since the late 1800s.
Like most stains used to detect lipids, Oil Red O is not a true special stain, since it
can’t form bonds with lipid components.
It is actually a pigment that functions as an oil-soluble colorant, and the technique
represents a physical method of staining.
For general use:
70% alcohol is an adequate solvent for Oil Red O and Sudan black.
Containers should always be kept covered except when the tissue is being placed
into and taken out of solution to prevent evaporation, particularly off their
alcoholic component.
Sudan Black Method for Lipids
Method: Fixation:
1. Sections from water to 50% and 70% alcohol. Formaldehyde calcium with
2. Stain for up to 2 hrs in saturated Sudan Black post-chroming.
B in 70% ethanol. Section:
3. Place in 70% alcohol for 5 seconds only to Unfixed cryostat section/ Frozen
remove excess surface dye. sections post fixed in formol calcium
Note: Longer periods will remove the color
4. Immerse in distilled water for 2 mins.
Results:
5. Wash in distilled water for 2 minutes.
Lipids Blue black
6. Counterstain with Mayer’s Carmalum for 2 ½
mins. Nuclei Red Optical view of an embryo stained with Sudan black B