In histotechnology, the word lipid refers to all fat and fat like, or fat containing substances.

includes triglycerides, fatty acids, lipoproteins and glycolipids.

Lipids or fats are generally classified into simple lipids, compound lipids, and derived lipids. They all have
a common property of solubility in organic solvents and insolubility in water.

1. Simple lipids (neutral fat) are esters of fatty acids with alcohols and are usually found in the body as
energy stores in adipose tissue. Triglycerides are esters of fatty acids with glycerol constituents, serving
as storage fats in animals with high solubility for certain non-ionic colored substances (lysochromes)
stainable by Sudan Black B, Sudan IV and Oil Red 0.

2. Compound lipids consist of a fatty acid, an alcohol and one or more other groups such as phosphorus
or nitrogen. They are generally found in the central nervous system.

a. Phospholipids are important components of cellular membranes particularly found in mitochondria

and nervous tissue elements and are readily stained by Sudan Black B and acid hematin.

b. Glycolipids are composed of fatty acids and hexoses, possessing characteristics of both lipids and
carbohydrates and are therefore stained by Sudan Black B and PAS techniques.

3. Derived lipids are fatty acids that are derived from hydrolysis of simple and compound lipids.
Examples are cholesterol, bile acids, sex hormones and adrenocortical hormones.

Adipose Tissue

Adipose tissue or fat is distributed throughout the body in distinct “white” and “brown” adipose tissue
depots. White adipose tissue (WAT) is largely composed of unilocular lipid-filled adipocytes that
specialize in lipid storage, whereas brown adipose tissue (BAT) is largely composed of multilocular
adipocytes that specialize in lipid burning. Fat cells appear as “signet rings” on H&E stain because large
lipid droplet displace the nucleus and remainder of the cytoplasm to the edge of the cell.

Lipid bodies, also named lipid droplets or adiposomes, are distributed in the 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 acid composition.

Cytoplasmic lipid droplets are neutral lipids, usually triacylglycerols or cholesteryl esters. Because lipid
bodies can be destroyed by drying or fixation and staining with alcohol based reagents, there are
consequently some methodological limitations to their study.

Lipids are difficult to demonstrate histologically because they dissolve in the solvents used for paraffin
processing and, to a lesser degree, in celloidin processing.

Frozen sections may be required in order to stain for lipids. Triglycerides are always completely removed
in properly cleared and paraffin infiltrated blocks.

Lipids are best demonstrated on cryostat sections of fresh unfixed tissue since there is no really good
fixative available. Formalin only preserves those lipids that are already more or less firmly bound to
proteins (such as lipofuscins and granules of leukocytes).
Lipochrome (lipofuscin) pigments are the breakdown products within cells from oxidation of lipids and
lipoproteins. They are the wear-and-tear pigments found most commonly in heart, liver, CNS, and
adrenal cortex.

Lipofuscins (lipochrome pigments) are PAS positive and variably acid fast. They stain with Ziehl-Neelsen.
In addition lipofuscin is Sudan black B and Sudan Red positive. Lipochrome can be demonstrated by
Schmorl's method which also stains for melanin. Lipochrome may also exhibit a strong orange auto
fluorescence in formalin-fixed, unstained paraffin sections.

Lipids present in fat embolism, fatty liver and atheroma may be fixed for staining in paraffin sections by
exposing the sections to an emulsion of linoleic acid and lecithin in 70% ethylene glycol at 56oC for 3
days.

These tissues are then treated with 2% chromic acid at 4°C for 24 h followed by 24 h in 5% sodium
bicarbonate, with appropriate rinsing between solutions. Paraffin sections of these tissues then stained
with a lipid-soluble dye such as Oil Red O. The demonstration of fat embolism with good quality tissue
detail is made practical by the method, which is convenient and inexpensive.

Phospholipids and neutral fats will be lost during routine dehydration and embedding unless they are
treated with potassium dichromate or osmic acid, which are the only agents that truly fix lipids. Formol-
calcium is the fixative of choice for lipid histochemistry, and is prepared by adding 2% calcium acetate to
10% formalin, neutral fats are still best demonstrated in frozen sections of fixed or unfixed tissue.

In general, histochemical techniques are the common methods of choice for demonstrating lipids in
tissue sections. These are usually complemented by other biochemical techniques and chromatography
for specific identification and possible quantification of the lipid demonstrated by microscopy.

Fat Stains and Sudan Dyes

Sudanophilia is the property of tissues to be stained with fat or oil-soluble dyes, regardless of the type
of dye used, due to their essential lipid nature. The staining is based on the greater physical solubility of
the dye in lipid substances than in 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 (neutral fat). Oil
soluble dyes are usually divided into the main groups:

1. Basic Aryl amines with very low water solubility:

a. Sudan Black B - most sensitive lipid stain known

b. Sudan Red VII B

2. B-Naphthols such as the original diazo dyes

a. Sudan III (C.I. No. 26100)
b. Sudan IV (Scharlach B) C.I. No. 26105 - staining fats with a more brilliant or deeper red color than
Sudan III which stains lipids orange-red.

Sudan dyes are a group of lipid soluble solvent dyes often called lysochromes. Sudan III was the first of
these dyes to be introduced in 1896, followed by Sudan IV (Scharlach R) in 1901. Sudan III is
predominantly used for staining triglycerides in animal tissues (frozen sections). With the use of certain
solvents, may also be used to stain some protein bound lipids in paraffin sections. The most sensitive
and versatile of all these dyes is Sudan Black B, which was introduced in 1935. Unlike other Sudan dyes,
Sudan Black B stains phospholipids as well as neutral fats. However, Sudan Black B 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.
The uptake of Sudan black B by partition from dilute solution is a specific test for lipid, but in normally
fixed tissue most of the structural lipid is ‘bound’ and is not accessible to the dye.

For frozen sections, cut tissues about 15 micra thick are usually stained with. Scharlach R or with Oil Red
0, which stains neutral fats and lipofuscin well. Oil Red O is used to demonstrate the presence of fat or
lipids in fresh, frozen tissue sections. Oil Red O is a fat-soluble diazo dye, and is 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 0 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 of their alcoholic component.

Sudan Black Method for Lipids (Suvarna 2013)

Fixation: Formaldehyde calcium with post-chroming
Section: Unfixed cryostat sections preferred, or frozen sections post-fixed in formol calcium
Method:
1. Sections from water to 50% and 70% alcohol.
2. Stain for up to 2 hours in saturated Sudan Black B in 70% ethanol.
3. Place in 70% alcohol for 5 seconds only to remove excess surface dye. (Longer periods will remove
the color).
4. Immerse in 50% alcohol for I minute.
5. Wash in distilled water for 2 minutes.
6. Counterstain with Mayer's carmalum for 2 1/2 minutes.
7. Wash in distilled water for 2 minutes.
8. Mount in aqueous mounting medium.

Results: Lipids blue black Nuclei Red

Sudan IV (Scharlach R) Stain for Lipids

Fixation: 10% Formalin
Sections: Frozen sections
Method: Collect 10 µ frozen sections in distilled water
1. Place in 50% alcohol for 1 minute.
2. Place in 70% alcohol for 1 minute.
3. Immerse in Sudan IV or Scharlach R (Oil Red 0 may be used) for 5-10 minutes.
4. Dip in 70% alcohol for 1minute (Longer periods will remove the color.).
5. Counterstain with Harris hematoxylin for 2 minutes.
6. Differentiate in 1% acid alcohol until only the nuclei are stained blue/ black under microscopic control.
7. "Blue" in tap water for 5 minutes.
8. Rinse in distilled water.
9. Mount sections on to slide from distilled water to an aqueous mounting medium.

Results: Lipids (mainly triglycerides) - red Nuclei - blue/black

NOTE: Scharlach R (Sudan IV) is the most commonly used stain, producing a rapid and permanent
coloration of lipid. The addition of benzoic acid to the staining solution materially intensifies the
resulting color and prevents deterioration. True fats stain intensely while cholesterol stains less
intensely.

Oil Red 0 Method in Dextrin (modified by Churukian 2000) Oil Red 0 is closely related to Sudan dyes and
was introduced in 1926 by French. It was popularized in 1943 by Lillie and Ashburn who advocated its
use as a 50 to 60% fresh aqueous dilution of a saturated 99% isopropanol stock solution. Oil Red 0 stains
neutral fats and lipofuscin well. The basis for staining lipids with an oil-soluble dye lies in its increased
solubility in fatty substances as opposed to the dye solvents which are used in routine tissue processing.
The choice of solvent for this reaction is also critical, since it must be able to extract excess dye without
dissolving the lipid to be stained- propylene glycol is the preferred solvent for this technique. The end
result is that fat and lipids in tissue sections stain bright red, and nuclei stain blue.

Fixation: Fresh frozen or frozen sections post-fixed in neutral buffered formalin

Sections: 5 µm sections mount on slides, air dry
Solutions:
Dextrin Solution Dextrin 1 gm
Distilled water 100 ml
Oil Red O stock solution –
Oil Red O 0.5gm
Isopropanol 100ml Dissolve the dye in isopropanol using gentle heat in water bath.
Oil Red O working solution
Stock Oil Red O solution 30ml
Dextrin 20ml Allow to stand for a day or more. Stable or months, filter before use.
Method:
1. Place slides directly into filtered 0.5% Oil Red O in dextrin. Stain for 20 minutes, rinse with running
water briefly.
2. Counterstain with Gill II hematoxylin for 20-30 seconds. Rinse with water, blue, coverslip with
aqueous mounting media.
Results Lipid red Nuclei blue

Osmic Acid Stain for Fat Osmium tetroxide (osmic acid) is not a dye but is an unstable oxide which is
reduced to a permanent black substance by unsaturated fats and fatty acids. It is used as a fixative for
electron microscopy and in histochemistry, and for demonstration of unsaturated fats, with the
disadvantage that other substances may also be stained simultaneously.
Lipids may be demonstrated by fixing the tissue in chrome-osmium solutions or by using frozen section.
Reduction of osmium by thiocarbohydrazide highly enhances lipid labeling. After formol/ glutaraldehyde
fixation much of the lipid in the tissues is ‘bound’ and does not take up osmium. It can be unmasked by a
saturated aqueous solution of thymol.

Fixation: 10% formalin

Section: Cryostat section
Method: 1. Collect frozen sections at l0 µ in distilled water.
2. Immerse in l % osmium tetroxide in the dark for 12-18 hours.
3. Wash in distilled water.
4. Wash thoroughly in tap water for 3 hours.
5. Counterstain if desired with l % safranin for l minute.
6. Rinse in distilled water.
7. Rinse in 70% alcohol.
8. Mount on to slides.
9. Blot dry.
10. Dehydrate rapidly, clear and mount.

Results: Nuclei yellow-orange Fats black

Nile Blue Sulfate Method for Fats

Nile blue sulfate is a dye capable of differentiating two lipid classes simultaneously by the action of its
two components: a red oxazone which dissolves neutral lipids, and a blue oxazine which is basic and
reacts with phospholipids and free fatty acids. Nile blue sulfate can be used as a preliminary indicator of
the type of lipid present in the tissue section. Nile red is an excellent stain that is present as a minor
component of commercial preparations of the non-fluorescent lipid stain Nile blue. Nile red is intensely
fluorescent and, if proper spectral conditions are chosen, it can serve as a sensitive vital stain for the
detection of cytoplasmic lipid droplets. Nile red 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
histochemical methods involve chemical reactions with specific groups, radicals or bonds in the lipid
molecule.

Free Fatty Acids The histochemical demonstration of free 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. Calcium and iron deposits
will also bind with copper, but can be distinguished from lipid by their persistence in a de-lipidized
control section. They can be extracted with either 1% hydrochloric acid (for calcium) or 5% oxalic acid
(for iron salts).

Cholesterol An enzymatic method introduced by Emeis et al in 1977 based on the production of H2O2
from free cholesterol by cholesterol oxidase.

Cerebrosides The sulfate esters of cerebrosides (sulfatides) are generally deposited in brain and other
organs of patients with sulfatide storage disease known as metachromatic leukodystrophy. 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. Staining with
cresyl violet produces a metachromatic orange color on sulfatides, in contrast to the orthochromatic
purple 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. The section is dehydrated
with acetone to eliminate the metachromasia induced by less acidic groups.

Gangliosides Neurons may have significant ganglioside storage giving rise to cells suggestive of
gangliosidoses, but storage may not be obvious particularly in subtypes without mental retardation. The
stored gangliosides are PAS (+), sudanophilic (+), and Luxol fast blue (+).
Gangliosides present in storage diseases 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 and sialic acid. A modified PAS method reduces the concentration of 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 gangliosides (the only glycolipids that
contain sialic acids) within neurons in Tay-Sach's disease.

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