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Staining of Lipids

Oil Red O
 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
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.
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
• 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.
• 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.
• Also suitable for cryostat sections
• Should be mounted on chrome-gelatin coated slides
(fixation causes the endogenous tissue proteins to lose their adhesive properties)
- 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.
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

7. Mount in aqueous mounting medium.

Sudan IV (Scharlach R) Stain for Lipids
Method: Fixation:
1. Place in 50% alcohol for 1 minute. 10% Formalin
2. Place in 70% alcohol for 1 minute. Sections:
3. Immerse in Sudan IV or Scharlach R (Oil Red O may Frozen Sections
be used) for 5-10 minutes
4. Dip in 70% alcohol for 1 minute (Longer periods will
remove the color). Results:
Lipids (Mainly Triglycerides) Red
5. Counterstain with Harris hematoxylin for 2 minutes.
Nuclei Blue/Black
6. Differentiate in 1% acid alcohol until only the nuclei
are stained blue or black under microscopic control.
7. “Blue” in top water for 5 minutes.
8. Rinse in distilled water.
9. Mount sections on to slide from distilled water to an
aqueous mounting medium.
Oil Red O Method in Dextrin
• Introduces in 1926 by French.
• Popularized in 1943 by Lillie and Ashburn who
advocated its use as a 50-60% fresh aqueous Fresh frozen/Frozen sections
dilution of saturated 99% isopropanol stock post-fixed in neutral buffered
solution. formalin
• Stains neutral fats and lipofuscin well. Sections:
Methods: 5µm sections mount on slides,
air dry.
1. Place slides directly into filtered 0.5% Oil Red O in
dextrin. Stain for 20 minutes, rinse with running
water brieflt. Results:
2. Counterstain with Gill II hematoxylin for 20-30 Lipid Red
seconds. Rinse with water, Blue, Cover slip with Nuclei Blue
aqueous mounting media.
Osmic acid Stain for Fat
• Not a dye but an unstable oxide which
reduced to a permanent black
1. Collect frozen sections at 10µ in
distilled water. substance by unsaturated fats and fatty
2. Immerse in 1% osmium tetroxide in acids.
the dark for 12-18 hours. • Used as a fixative for electron
3. Wash in distilled water. microscopy and histochemistry and for
4. Wash thoroughly in tap water for 3 demonstration of unsaturated fats.
5. Counterstain if desired with 1% Fixation:
Safranin for 1 minute. 10% formalin
6. Rinse in distilled water. Section:
7. Rinse in 70% alcohol. Cryostat section
8. Mount on to slides. Results:
9. Blot Dry. Nuclei Yellow-orange
Fats Black
10.Dehydrate rapidly, clear and mount.
Nile Blue Sulfate Method for fats
• A dye that is capable of differentiating two lipid classes simultaneously by the
action of its two components:
 Red oxazone –which dissolves neutral lipids
 Blue Oxazone –which is basic and reacts with phospholipids and free fatty
• Preliminary indicator of the type of lipid present in the tissue sections.
Nile Red
• Excellent stain that is present as a minor component of commercial preparations of
the non-floursecent lipid stain Nile blue.
• Intensely fluorescent and can serve as sensitive vital stain for detection of
cytoplasmic lipid droplets if proper spectral conditions are chosen.
• Provides resolution of cytoplasmic lipid droplets in tissues equal to, if not better
than, that obtained with the non-fluorescent dye oil red O.
Histochemical Methods
- Involves chemical reactions with specific groups, radicals or
bonds in the lipid molecule.
Free Fatty Acids
The Histochemical demonstration of free fatty acids is based on the
observation that free fatty acids bind heavy metal ions such as copper
to form soaps which can then be stained with Weigert’s lithium
hematoxylin, dimethylaminobenzidine rhodamine, or rubeanic acid.
Note: Calcium and Iron deposites will also bind with copper.
De-lipidized control section
• 1% hydrochloric acid (for Calcium)
• 5% oxalic acid (for Iron salts)
• Most of the earlier methods for demonstrating cholesterol were ineffective unless
cholesterol had been oxidized, either chemically with ferric salts or by long exposure to
atmospheric oxygen.
• 1977 - Emeis et al introduce an enzymatic method based on the production of H2O2
from free cholesterol by cholesterol oxidase.
• It involves a two stage procedure:
1. Esterified cholesterol is initially hydrolyzed to its free sterol by means of cholesterol
2. Cholesterol is then oxidized by cholesterol oxidase to release hydrogen peroxide, which
reacts simultaneously with diaminobenidine to produce an insoluble brown polymer at
the site off cholesterol.

Cholesterol esters can be demonstrated as cholesterol after hydrolysis by cholesterol

ester hydrolase and has been detected with the use off enzyme cholesterol esterase.
These methods are of limited practical use :
• Reagents are expensive
• Too complex for routine light microscopy
Metachromatic leukodystrophy - a sulfatide storage disease in which the sulfate ester of cerebrosides
(sulfatides) are generally deposited in brain and other organs.
Cerebrosides and related lipids are stained by the Periodic Acid Schiff (PAS) method, designed to stain
mucopolysaccharides, and can be distinguished from glycogen by removal with diastase.
Cresyl violet - produces a metachromatic orange color of other, less acidic myelin lipids.
Toluidine blue - is a standard metachromatic dye for acidic polymers, and imparts a yellow
brown or purplish color to sulfatide deposits.
To eliminate the metachromasia induced by less acidic groups, the section is
dehydrated with acetone.

Neurons may have significant ganglioside storage giving rise to

cells suggestive off GANGLIOSIDOSES, but storage may not be
obvious particularly in subtypes without mental retardation.
Stored ganglioside are:
• PAS (+)
• Sudnaophilic (+)
• Luxol fast blue (+)
Gangliosides present in storage disease like Tay-Sach’s disease
and Gm1 gangliosidosis are stained with conventional PAS method.
They are distinguished from other glycolipids by their constituents,
Neuraminic acid & Sialic acid.
A modified PAS method reduces the concentration o the oxidizing
agent (Periodate) from 1 to 0.01% to stain sialo-groups that are oxidized
more rapidly than other sugar glycoside.
This modified PAS stain has been used to demonstrate ganglioside (the
only glycolipids that contains sialic acids) within neurons in Tay-Sach’s