Department Of Anatomy

Faculty Of Basic Medical Sciences,

Enugu State University College of
Medicine, Enugu

HISTOCHEMISTRY OF LIPID

Dr. Ezugwu, N.S

NOTE: This study guide is not

for sale
GOALS

• To understand the basic classification and chemistry of lipids

• To describe the different Histophysical and histochemical methods

of identifying lipids.
• To appreciate lipid immunohistochemistry
• To discuss the clinical applications of lipid Histochemistry.
 OUTLINE

• INTRODUCTION

• CHEMISTRY AND CLASSIFICATION OF LIPIDS

• FIXATION FOR LIPID HISTOCHEMISTRY

• CONTROL SECTIONS

• HISTOPHYSICAL METHODS

• HISTOCHEMICAL METHODS

• LIPID IMMUNOHISTOCHEMISTRY

• APPLICATION OF LIPID HISTOCHEMISTRY

 LIPID HISTOCHEMISTRY

WHAT IS HISTOCHEMISTRY?
Histochemistry is the science concerned with the identification and
distribution of the chemical constituents of tissues by means of stains,
indicators, and microscopy which is based on chemical reaction between
cell components and stains. The end product of the reaction is permanent
colored stains that can be viewed under the microscope.

LIPIDS
Lipids are referred to any one of a group of naturally occurring fats and
fat like substances characterized by their insolubility in water (Bancroft).
Histochemical methods are the most common methods of choice for
properly demonstrating lipids in tissue section. According to Gomori
1952, lipid is a term used to denote a large group of miscellaneous
chemical substances classified together, for histochemical purposes only,
by their solubility properties. These properties are insolubility in water
and solubility in several or all of the so-called "fat solvents" (alcohol,
ether, chloroform, benzene, pyridine, acetone, etc.). An immense variety
of lipid substances occur in the animal and plant kingdoms.

CHEMISTRY OF LIPIDS
The common feature of lipids is that they are all esters of moderate to
long chain fatty acids, acid or base catalyzed yields the component fatty
acids.
Fatty acids are mainly straight chain aliphatic mono carboxylic acids
ranging in chain length from 4 -24 carbon atom (R-COOH).
The carbon chain may attach to functional groups containing oxygen,
halogen, nitrogen and sulfur.
If the fatty acid contains a double bond, there is a possibility of either Cis
or Trans geometric isomerism.
Cis double bond cause the fatty acid chain to bend, an effect that is
compounded with more double bond in the chain.
Most naturally occurring fatty acids are the Cis configuration.
Fatty acids can be saturated or unsaturated. Saturated have a higher
melting point than unsaturated fatty acid of corresponding size.

SATURATED FATTY ACID.

Saturated fatty acid belong to the group of acetic acid series with general

formula . They contain up to 8 carbons and are liquid at room

temperature.
Systemic name ends at anoic. E.g. Palmatic,Stearic,Arachidinic ,Lauric.

UNSATURATED FATTY ACIDS.

Unsaturated fatty acids contain double bonds, systematic naming ends at
enoic. Unstable and reactive due to the presence of double bond, the
double bond gives rise to the possibility of geometric isomerism,
E.G. oleic(oleic is the most abundant fatty acids in nature forming about
50% of total fatty acids in fats, found naturally in existing fats.

INDEPENSABLE FATTY ACIDS (must be gotten from diets, these

includes Linoleic acid and Arachidonic acid
Structures of some common fatty acids

CLASSIFICATION OF LIPIDS
Lipids can be classified based on
 The presence or absence of glycerol
 The combination with other compounds
 The presence or absence of additional group.

Classification based on presence or absence of glycerol

Fatty acid
Lipids containing glycerol
 Neutral fat (triglycerides)
  Phospholipids
 Cardiolipin
 Plasmalogen
Lipids without glycerol
 Sphingolipid
 Waxes
 Terpenes
 Steroids
 Prostaglandin
Lipid combined with other compounds
 Lipoproteins
 Glycolipids

Classification Based On Presence/Absence of Additional Group

1. Simple lipids (fats and waxes)

2. Complex lipids (esters of fatty acid containing group in addition to
alcohol and fatty acid. Examples include:
• Phospholipids (fatty acid, alcohol and phosphoric group)
• Sphingophospholipid (sphingosine)
• Glycolipids (glycosphingolipid) lipid containing fatty acids,
sphingosine and carbohydrate
• Other complex lipids are sulfolipids, aminolipid, lipoprotein
3. Precursor or derived lipids: These include fatty acids, glycerol,
steroids, and other alcohols like fatty aldehyde, ketone bodies,
hydrocarbon, and lipids soluble vitamins.
Classification of Lipids (Gomori 1952)
A. Paraffin (petrolatum).
B. Isoprene derivatives (Carotenoids). This group includes carotene,
vitamin A, visual purple, and some of the pigments of crustaceans.
C. Fatty acids and their derivatives.

a. Fatty acids.
b. Soaps
c. Triglycerides (neutral fats).
d. Waxes (long-chained alcohol esters of fatty acids).
e. Phosphatids(lecithin,cephalin,plamalogens spingomyelin)

D. Cerebrosides ( Sphingosine galacatoside or glucoside)

E. Lipid peroxides.

F. Steroids( cholesterol, cholesterol esters ,steroid hormones)

 Table: Classification of Lipids

FIXATION AND MICROTOMY FOR LIPIDS

Fixation helps lipids withstand effects of reagents

• Osmium tetraoxide and chromic acid (fix lipids but alter reactivity)
• Formal calcium (2% calcium acetate + 10% formalin) always a
choice fixative
Fresh frozen material will give the best results but where fixation is
helpful, post fixation of cryostat sections will suffice (Bancroft and
Gamble, 2007).

MICROTOMY
Cryostat sections.
• Few drops of water and unfixed tissue are put on the cutting edge
of cryostat and are frozen.
• sections are cut frozen in cryostat
• Sections are picked with slides and used for staining.
• Formal-calcium fixed blocks of tissue are also suitable for cryostat.

CONTROL SECTIONS
Control sections are important in lipid Histochemistry:

TYPES OF CONTROL SECTIONS

1. NEGATIVE CONTROLS (Lipid extraction)

a. Keilig’s (1944) Extraction Method
• Blocks of unfixed tissue 2-3mm thick are extracted using the
solvents.
• Three changes of solvents are used.
First and second for 3 hours
Third for 12 hours

KEILIG’S EXTRACTION METHOD

LIPID EXTRACTED SOLVENT

Glycerides, cholesterol and its Cold acetone
esters

Cerebrosides Hot acetone

Lecithin and Cephalins Hot ether

All lipids Hot chloroform-methanol (equal

parts)

After extraction, the blocks are taken down to water through descending
grade of alcohol and frozen sections are cut and stained.

2. Baker’s (1944) pyridine extraction method:

Fixation
Weak bouin’s fluid;
Saturated aqueous picric acid 50ml
Commercial formalin 10ml
Glacial acetic acid 5ml
Distilled water 35ml

Sections
10µ frozen sections
 TECHNIQUE
1. Fix small portions of tissue in weak Bouin for 20 hrs
2. Wash in alcohol to remove picric acid
3. Extract in pyridine at room temperature for 30 mins
4. Extract in pyridine at 60ºC for 24 hours
5. Wash in running water for 2 hours
6. Treat with dichromate- calcium

Result: All lipids removed

POSITIVE CONTROL
• Can be obtained from known cases of lipid storage disorders
• Commercially obtained pure samples can be applied to filter paper
and treated as tissue section in the relevant technique.

IDENTIFICATION OF LIPIDS
The actual procedures of demonstrating lipids are divided into two
groups-physical and chemical methods (Gomori 1952; Bancroft,).

FAT STAINS AND SUDAN DYES

Staining with oil-soluble dyes (fat stains and Sudan dyes).-

 There is a large number of oil-soluble dyes known, many of which are
suitable for histological purposes (Sudan III and IV, Sudan black B,
Oil red O, Blue B.Z.L., Nile blue; etc.).
With the exception of Sudan black B, which is an excellent stain for
phosphatides, all the dyes mentioned above stain triglycerides and
fatty acids in a much darker shade than phospholipids. The latter may
actually remain practically unstained. When using Nile blue, it should
be remembered that it is not a specific lipid stain.
While the Sudan stains lipids and nothing else, not all structures
stained either red or blue by Nile blue are of a lipid nature. The two
most important features of lipid substances affecting their stainability
by dyes are their melting points and the presence or absence of
hydrophilic groups (especially if the dye is applied in an aqueous
solution).
Sudan staining is a simple physical process as the dyes are more
soluble in tissue fats than in their dye solvent. Only lipids that are
liquid or semi-liquid at staining temperature will be stained since the
adsorbing power of fats are related to temperature, dye concentration,
physical state and melting point (max. dye uptake occurs at melting
point).

OIL RED O FOR THE DEMONSTRATION OF FATS

PRINCIPLE
• suitable for tissue containing lipids in tiny droplets
• cryostat sections on fresh or fixed tissue are used.
• stain lipids by being more soluble in the lipids than in their
solvents.
REAGENTS
(A) OIL RED O STOCK SOLUTION
• Oil red O 0.5 g
• Isopropanol 500 mL
• Warm the dye and alcohol in a long necked flask over a water bath
at 56ºC for 30 - 60 mins. Cool.
OIL RED O WORKING SOLUTION
• Stock solution 30 mL
• Distilled water 20 mL
• Mix well and stand for 5 to 10 mins. Filter. Use same day.
(B) FORMAL CALCIUM
2% calcium acetate+10% formalin
(C) HARRIS OR MAYER'S HAEMATOXYLIN
• PROCEDURE
1. Stain in working solution - 5 - 10 mins in a closed container.
2. Wash well in water but gently (sections tend to float).
3. Harris Haematoxylin - 1/2 - 1 min or Mayer's Haematoxylin 3
minutes.
4. Blue in tap water.
5. Rinse in distilled water.
6. Mount in aqueous mounting medium.
• RESULTS
Triglycerides (neutral fats) Deep orange red
Nuclei Blue
Photomicrograph of liver section, demonstrating fats stained deep
red and Nuclei blue by oil red O stain. x400
Photo credit: Zuhal, 2005.

SUDAN BLACK B METHOD

PRINCIPLE
• Sudan black B stains phospholipids as well as neutral fats.
• It fails to stain crystalline cholesterol and lecithins.
• These drawbacks are overcome if bromine pre-treatment is
included in the staining procedure.
• Bromine converts crystalline cholesterol to derivatives that are oily
at room temperature and hence permeable to the sudan dye.

Standard Sudan black B method for fats and phospholipids

Fixation and sections

 Cryostat sections post-fixed in formal calcium; short
fixed frozen sections; unfixed cryostat sections
(preferred)
 Method
 Rinse sections in 70% ethanol
 Stain for up to 2 hours in saturated sudan black B in
70% ethanol
 Rinse in 70% ethanol to remove excess surface dye
and wash in tap water
 Counter stain nuclei with Kernechtrot for 2-5 minutes
 Wash well and mount in glycerin jelly
Results
 Unsaturated esters and triglycerides ---- blue-black
 Some phospholipids may appear gray

Micrograph of red muscle fibre: Lipid droplet stained blue black by

Sudan B with a dark triangular area of intramuscular adipose tissue.
PHOTO CREDIT: Histoserv Inc, 2015
BROMINE SUDAN BLACK METHOD FOR LIPIDS (Bayliss and
Adams, 1972).
Fixation and sections

• Cryostat sections post-fixed for one hour in formal calcium

• Short fixed frozen sections
Method
1. Mount sections unto slides and allow to dry
2. Immerse sections in 2.5% aqueous bromine for 30 minutes at room
temp., inside a fume hood.
3. Wash in water and treat with 0.5% sodium metabisulfite for 1
minute to remove excess bromine.
4. Wash thoroughly in distilled water and treat, together with an un-
brominated section, for the standard Sudan black method.

BROMINE –ACETONE SUDAN BLACK METHOD FOR

PHOSPHOLIPIDS ( Bayiss High, 1981)
Method
1. Treat sections with 2.5% aqueous bromine for 30 minutes at room
temperature, inside a fume cupboard.
2. Wash well and remove excess bromine with 0.5% metabisulfite for
one minute.
3. Wash thoroughly and allow slides to dry
4. Extract neutral lipids with anhydrous acetone for 20 minutes at
48ºC
5. Proceed with the standard Sudan black method outlined above
Results
Phospholipids ---Gray
Sphingomyelins ---- Bronze in polarized light

NILE BLUE SULFATE METHOD FOR ACIDIC AND

NEUTRAL LIPIDS ( After Cain, 1947)
PRINCIPLE
Nile blue sulphate serves as a preliminary test to indicate the
distribution of neutral versus acid lipids.

Nile blue sulphate comprises two components;

• a red oxazone which dissolves in neutral lipids
• a blue oxazine which is basic and reacts with
phospholipids and free fatty acids.

Other methods of staining phospholipids are the bromine-acetone Sudan

black method and the Acetone-Nile blue sulfate method.

FIXATION AND SECTION

6 - 8 µm frozen section post fixed for one hour in formal-calcium.
6 - 8 µm Cryostat sections on tissue fixed in 10 % neutral buffered
formalin.
CONTROL
Neutral fat subcutaneous tissue or fatty liver.
• REAGENTS
(A) NILE BLUE SULPHATE SOLUTION
• Add 10mLs of 1% Sulphuric Acid to 200mLs of 1% Nile Blue
Sulphate in water.
• FIXATION AND SECTION
• 6 - 8 µm frozen section post fixed for one hour in formal-
calcium.
6 - 8 µm Cryostat sections on tissue fixed in 10 % neutral
buffered formalin.
• CONTROL
Neutral fat subcutaneous tissue or fatty liver.

• REAGENTS
(A) NILE BLUE SULPHATE SOLUTION
• Add 10mLs of 1% Sulphuric Acid to 200mLs of 1% Nile Blue
Sulphate in water

• Boil under reflux for 4 hours.

• (B) FORMAL CALCIUM
• 2% calcium acetate+10% formalin

(C) 1% CHLOROFORM-WASHED METHYL GREEN

•
• PROCEDURE
1. Cut frozen sections and fix for 1 hour in formal calcium.
2. Stain in Nile Blue Sulphate at 60ºC for 30 mins.
3. Differentiate in 1% acetic acid for 1 - 2 mins.
4. Wash well and counterstain nuclei with 1% chloroform-washed
 methyl green 5 mins.
5 Wash well and mount in glycerine jelly.
RESULTS
• Unsaturated hydrophobic lipids Pink
• Free fatty acids Pink to blue
• Phospholipids Blue

ACETONE-NILE BLUE SULFHATE METHOD FOR

PHOSPHOLIPIDS (Dunnigan, 1968).
Fixation and sections
• As in methods above
Method
1. Mount sections unto slide and leave until dry
2. Treat with 1M HCl for 1 hour to convert to calcium soaps of the
free fatty acids.
3. Wash and dry sections
4. Extract with acetone at 4ºC for 20 minutes
5. Dry sections rapidly and proceed as for previous method but
counterstain nuclei with kernechtrot instead of methyl green.
Results
Phospholipids -------- blue

HISTOCHEMICAL METHODS

Once the presence of lipid in a specimen has been found, specific

histochemical procedures to determine its identity can be applied.
 Unlike fat stains, histochemical methods involve chemical reactions
with specific groups, radicals or bonds in the lipid molecule, taking
advantage of any unique configuration that enables discrimination
between close members of a lipid class although the value of this method
can be limited by interference from non-lipid substances, hence the need
for control sections.

Table showing some histochemical methods for demonstration of

specific lipids:

Histochemical Lipid Fixation Result

Techniques Demonstrated

Schultz method Cholesterol and Formal saline Green

its esters

Filipin method Free cholesterol Post fixed Silvery

formal -calcium fluorescence

Ferric All formal calcium Dark green

Heamatoxylin phospholipids

Osmium Un-saturated Unfixed or SF Brown – black

tetroxide fatty acids
Reduction
OTAN Phospholipids Un fixed or SF Orange – Red

NAOH- OTAN Sphingomyelin Un fixed or SF Orange – red

Modified PAS Cerebrosides Formal calcium Magenta

reaction ( reddish–
purple)

Boro – Hydride Gangliosides Formal calcium Red

periodate Schiff

Plasma reaction Plasmologens Unfixed Magenta

Schultz Method for Cholesterol and Cholesterol Esters.

This method depends on ability of iron alum to convert cholesterol to

oxychlosterol which is the converted to 7- hydroxyl cholesterol when
equal volume of concentrated sulphuric acid and glacial acetic acid are
added to it through development of blue, purple colors. It is the final
green color that is diagnostic of cholesterol.

Method:
1. Attach frozen sections of 10 – 15 um thickness to slides
2. Apply about 2 drops of concentrated sulphric acid onto sections—5
to 10secs
 3. Apply about 3 to 4 drops of acetic anhydride onto the sections
4. Drain the acetic anhydride from the sections and wash with acetic
anhydride
5. Mount in acetic anhydride and examine immediately
Result
 Cholesterol and cholesterol esters --------- red–violet which
changes to green.

Cryostat sections of Gaddi goat showing moderate cholesterol

reaction (green colour)

Filipin method for free cholesterol

Fixation and sections

 Cryostat sections, post-fixed; frozen sections of fixed tissue
Preparation of reagent
Stock solution
 2.5 mg filipin in 1 ml dimethylformamide
Staining solution
  Filipin stock solution ---- 0.2ml
 PBS (phosphate buffered saline) ---- 10ml
Method
1. Wash sections in PBS
2. Stain for 30 minutes in Filipin solution
3. Wash in PBS (x2)
4. Mount in PBS or glycerin jelly
5. Examine by fluorescence microscopy
Results
 Free cholesterol shows silvery fluorescence

Other histochemical methods of demonstrating cholesterol include

percholoric acid-Naphthoquinone (PAN) method and the digitonin-PAN
method.

OSMIUM TETROXIDE METHOD FOR UNSATURATED LIPIDS

Fixation
 Preferably unfixed cryostat sections; otherwise use short-fixed
frozen sections
Method
1. Immerse sections in 1% aqueous OsO 4 for 1 hour at room
temperature
2. Wash well in distilled water and mount in glycerin jelly
Results
 Unsaturated lipids are stained brown to black
 Saturated lipids and free cholesterol do not react
Micrograph of frontal section of veliger larva

OSMIUM TETROXIDE α NAPHTHYLAMINE (OTAN) FOR

PHOSPHOLIPIDS

• For differentiating hydrophilic phospholipids from hydrophobic

lipids.
• Examples of hydrophilic phospholipids are sphingomyelin,
cerebrosides and cephalins.
Examples of hydrophobic lipids are cholesterol esters, fatty acids, and
triglycerides
Solutions:
1% ossium tetroxide --------1 ml
1% potassium chlorate -----3ml
α—napthylamine solution – add α—napthylamine solution to water
heated to 40oc, until is saturated and filter.

Method
• Float fixes frozen sections in ossium tetroxide solution for 18 hours
 • Wash sections in distilled water for 10 minutes and mount on glass
slides
• Transfer slides to α-napthylamine solution at 37o c-----20mins
• wash in distilled water
• counter stain in 1% alcain blue in 3% acetic acid ----------15-
30mins (optional)
• wash in distilled water
• mount in glycerine jelly
Result
• unsaturated lipids( triglycerides and fatty acids)--------Black
• Phospholipids --------------- orange - Red

SODIUM HYDROXIDE (NAOH)-OTAN FOR SPHINGOMYELIN

This method is based on the fact that sphingomyelin is not removed by

alkaline solutions, while phospholipids are removed by treatment with
alkaline solutions.
• if a tissue is treated with 2N sodium hydroxide and subsequently
stained with OTAN, and only sphingomyelin will be stained

Method:

• Treat sections with 2M sodium hydroxide at 37oc for 60mins

• Rinse in water
• Rinse in 1% acetic acid
• Rinse in water
• Transfer slide to ossium tetroxide solution for 18 hours and
continue with the OTAN method.

Result:
 • sphingomyelin----- orange to red

PLASMA REACTION FOR PLASMALOGEN PHOSPHOLIPIDS

This method depends on the ability of 1% mercuric chloride to expose

aldehyde groups in plasmalogens and acetal phospholipids.
These then reacts with schiff’s reagent to give a red colour.
Excessive exposure of tissue to mecuric chloride and schiff’s reagent
will give a false positive result.
The tissue must not be fixed.

Fixation
 Unfixed cryostat sections stained as soon as possible after cutting
Method
1. Air-dry duplicate sections onto separate slides
2. Hydrolyze one section in 2% mercuric chloride for 10 mins
3. Wash thoroughly in distilled water, three changes
4. Stain both sections with Schiff’s reagent for 10 mins
 5. Rinse in distilled water and wash in running tap water for 10 mins
to develop the colour
6. Counterstain nuclei with Mayer’s hematoxylin for 3 mins
7. Wash again in tap water, rinse in distilled water and mount sections
in glycerin jelly
Results
 Plasmalogen phospholipids (mainly phosphatidyl ethanolamine)
---- magenta

Ferric Hematoxylin (FeH) method for phospholipids

Fixation
 Ideally unfixed cryostat sections; otherwise short-fixed frozen
sections
Preparation of reagent
Solution A
 Distilled water ---- 298 ml
 Concentrated HCl ---- 2 ml
 FeCl3 6H2O ---- 2.5g
 FeSO4 7H2O ---- 4.5g
This solution can be stored
Solution B
 Distilled water ---- 10 ml
  Hematoxylin ---- 0.1g
 Dissolve by gentle heat, this solution must be fresh
Working Solution
 Mix three parts of (A) with one part of (B) and use within the hour
Method
1. Treat duplicate sections as follows:
a. Extract one section in chloroform: methanol (2:1) for 1 hour
at room temperature
b. Extract the other with dry acetone at 4oC for 15 mins
2. Fix both sections in formal calcium for 30 mins
3. Rinse in distilled water
4. Stain in the ferric hematoxylin solution for 7 minutes
5. Wash in distilled water
6. Dip several times in 0.2% HCl
7. Wash in tap water
8. Dehydrate in acetone, clear in xylene and mount in DPX
Results:Phospholipids ---- blue ,Nuclei ---- blue

CEREBROSIDES

This can be demonstrated by reaction specific for hexose

molecule.
Stained by PAS method.

Periodic acid coverts the 1-2 glycol group in hexose –molecule to

Schiff stainable aldehyde.
 PAS reaction is designed to stain hexoses, the distinction between
glycolipids and non –lipids hexoses depend upon comparism with a
de-lipidized control section

Modified PAS reaction for Cerebroside

Fixation
 Cryostat sections post-fixed in formal calcium; fixed frozen
sections.
Preparation of reagent
Performic acid
 98% formic acid ---- 45 ml
 100 vol hydrogen peroxide ---- 4.5 ml
 Concentrated H2SO4 ---- 0.5 ml
Prepare an hour before use and stir occasionally with a glass rod, inside a
fume hood, to release bubbles of gas from the solution
Method
1. Mount duplicate sections onto separate slides and extract one of
these with chloroform methanol (2:!v/v) for 1 hour at room
temperature.
2. Deaminate both sections in 10% aqueous chloramine T for 1 hour
at 37oC
3. Wash slides vigorously and as rapidly as possible, one at a time in
a large volume of water before transferring them immediately to
performic acid for 10 mins. The washing must be swift yet
thorough to avoid swelling and detachment of sections from slides
4. Wash well in distilled water
 5. Treat in a filtered saturated solution of 2,4-dinitrophenyl hydrazine
in M HCl at 4oC for 2 hours
6. Wash well in water
7. Treat with 0.5% periodic acid for 10 mins
8. Wash in distilled water
9. Stain in Schiff’s reagent for 15 mins
10.Rinse in distilled water and wash in taps water for 15 mins to
develop colour
11.Counterstain nuclei with Mayer’s hematoxylin or Corazzi’s
hematoxylin if wished
12.Wash in tap water, distilled water and finally mount sections in
glycerin jelly
Results
 Cerebroside ---- magenta
Indicated by the difference in staining intensity between the two sections

Photomicrograph of brain tissue showing reddish-purple stained

Cerebrosides.
PHOTO CREDIT: Histoserv Inc, 2015
GANGLIOSIDES

Gangliosides are differentiated from other glycolipids on account

of their constituent neuraminic acid and sailic acid.

Conventional PAS method has been proven to be the most practical

and rewarding approach for detecting gangliosides. (Bankcroft,
2008).

Histochemical method of gangliosides involves the use of routine

PAS reaction, followed below (Boro-Hydride Periodate Schiff
method)

Borohydride-Periodate-Schiff (BHPS) method for Gangliosides

Fixation
 Cryostat sections post-fixed in formal calcium; frozen sections of
fixed tissue
Method
1. Destroy existing aldehyde groups (endogenous or from formalin or
glutaraldehyde fixation) by reduction with 0.1 M (0.38%) sodium
Borohydride in 1% disodium hydrogen phosphate for 1 hour at
room temperature
2. Wash thoroughly in distilled water
3. Oxidize with 1.2 mM (0.03%) sodium periodate for 30 minutes at
room temperature
4. Wash twice for 5 mins each time in distilled water
5. Stain with Schiff’s reagent for 10 minutes
 6. Rinse in distilled water and wash well in tap water
7. Counterstain with Mayer’s hematoxylin or Corazzi’s hematoxylin
8. Blue in tap water, rinses in distilled water, then mount sections in
glycerin jelly

Results
 Gangliosides ---- red
 Nuclei ---- blue

Gold Hydroxamic method for Phosphoglycerides

Fixation
 Cryostat sections post-fixed for 1 hour in formal calcium; short
fixed frozen sections.
Preparation of reagents
Hydroxylamine solution
 Hydroxylamine hydrochloride ----2.5g
 Sodium hydroxide ---- 6g
 Distilled water ---- 100ml
Silver solution
 Silver nitrate ---- 0.1g
  Ammonium nitrate ---- 0.2g
 Distilled water ---- 100ml
Adjust pH to 7.8 with dilute sodium hydroxide

Method
1. Treat free floating frozen sections or slide mounted cryostat section
in the hydroxylamine solution for 20 mins
2. Wash the sections thoroughly in three changes of distilled water, 5
minutes each change
3. Immerse in the silver solution for 2 hours at room temperature
4. Wash well, rinse in 1% acetic acid and again in distilled water
5. Tone the sections for ten minutes with 0.2% yellow gold chloride
6. Rinse in water
7. Remove any unreduced silver by immersing in 5% sodium
thiosulfate for 5 mins
8. Wash well and mount section onto slides
9. Counterstain nuclei with 1% methyl green for 5 mins
10.Mout in glycerin jelly or dehydrate, clear and mount sections in
Canada balsam or synthetic resin

Results
 Phosphoglycerides (lecithins and cephalins) ---- purple
LIPID IMMUNOHISTOCHEMISTRY

• Majority of lipids are not antigenic

• Only gangliosides and galactocerebrosides can be demonstrated

immunohistochemically.
Conventional histochemical techniques best localizes and identifies lipids
microscopically

APPLICATION OF LIPID HISTOCHEMISTRY IN PATHOLOGY

Lipid Histochemistry can be applied in the following disorders:

A) NERVOUS SYSTEM DISORDERS

B) CARDIOVASCULAR DISORDERS

C) SKELETAL MUSCLE DISEASES

NERVOUS SYSTEM DISORDERS:

A. METABOLIC

B. B) NON METABOLIC

METABOLIC DISORDERS OF NERVOUS SYSTEM

A group of rare diseases that were thought to be degenerative.

Inherited disorders due to deficiencies of certain enzymes or co-
enzymes in the lysosome involved in lipid metabolism.
Leading to accumulation of relevant lipids or its metabolite at the
defective stage of metabolism
3 major categories:
A) Neurolipidosis : Lipids accumulate in the neurons
Example: Batten’s disease ( lipofuscinosis)

B)Leucodystrophies : lipid accumulation within the mononuclear

phagocytes in the white matter of the brain
.
• metachromatic leucodystrophies (sulphatides)
• krabbes leucodystrophy(galactocerebroside

C) Visceral lipidoses: lipids accumulate predominantly in the

mononuclear phagocytes.

DIAGNOSIS:
• Histochemical techniques only or
• Histochemical techniques reinforced by electron microscopy, thin
layer chromatography and enzymology
OTHER NERVOUS SYSTEM DISEASES
 • Demyelination of nerves : example- multiple sclerosis

• Myelin acts as insulator for nerve axons

• Helps transmit nerve signals faster

• Contains lipids, a compacted cell membrane a lamella structure of

free cholesterol, glycolipids and phospholipids (Hydrophilic).

DEMYELINATION OF NERVES
• In demyelination

a) Myelin structure is damaged

b) Fatty droplets are released

Cholesterol esters aggregate in macrophages (Pathognomic)

To indicate demyelination

• A) Frozen sections are required

B) Dichromate acid hematin / oil red combination recommended –

Good colour contrast between blue myelin and red stained esters within
macrophages.

C) Osmium tetraoxide –α naphthylamine (OTAN) – specific

 D) Sudan black B:
• normal myelin (gray)
• degenerating myelin (intense blue)

PARKINSONS DISEASE

• A Neuro-degenerative disease that is characterized by tremor rigidity

and akinesia ( slowing of movement)

• Progressive brain cell degeneration with accumulation of neuronal

Eosinophilic inclusion bodies – Lewy Bodies (pathognomic)

• Lewy bodies contain sphingomyelin Demonstrating the

sphingomyelins in lewy bodies:

• NaOH –OTAN procedure

• Result: orange- red color for sphingomyelin.

CARDIOVASCULAR DISEASES

A) ARTHEROSCLEROSIS: Deposition of cholesterol in the arterial

wall.
Cholesterol is present in free and ester forms
Method:
Stain arterial section with oil red o
View under polarized light
Result:
 Red stained fatty acids
Unstained birefringent crystalline cholesterol

B) CARDIOMYOPATHIES:

Lipid accumulation in heart muscles.

• Triglycerides are commonly implicated.
• Methods: Any fat stain

C) CHRONIC ANAEMIA (Thrush breast heart)

Triglycerides implicated too

D) Brown atrophy of heart muscles

Lipofuscin implicated

SKELETAL MUSCLE DISORDERS

Any excess neutral fat in muscle biopsy can be detected by

• Oil red O
• Sudan Black can also be used.

OTHER APPLICATIONS OF LIPID HISTOCHEMISTRY

• It is used in the diagnosis of steatosis of the liver (i.e., fatty liver)
• In histopathology, it is employed in the diagnosis of chronic
myelogenous leukemia
• It is used in the detection of wide spread cholesterol in mastoid
cholesterol granuloma
• Found useful in the investigation of lipid metabolic disorders e.g.,
Cerebrotendinous xanthomatosis, Sitosterolemia.
 • In diagnostic pathology it is used to investigate the presence of
demyelinating diseases of the central nervous system
• In pathology > detection and characterization of lipids in diseases
such as cardiomyopathies, brown atrophy in heart muscle,
leucodystrophies

HISTO-PHYSICAL METHODS:

TABLE SHOWING HISTOPHYSICAL METHODS OF LIPID

DEMONSTRATION

Histophysical Class of lipid Fixation Result

method demonstrated

Fluorescence Simple and Formalin or Silvery-white

microscopy compound lipids formal-calcium fluorescence

Polarized Cholesterol esters Formalin or birefringence

microscopy and some compound formal-calcium
lipids

Oil red O Neutral fats Formal calcium Neutral fats–

(triglycerides deep red
Nuclei ----- blue

Standard Sudan Unsaturated esters Formal calcium Triglycerides –

Black B and triglycerides, or none blue-black
some phospholipids Phospholipids --
gray

Bromine-acetone Phospholipids Formal calcium Phospholipids—

Sudan black gray
Sphingomyelins
– bronze in
polarized light

Nile blue sulfate Acidic and neutral Formal calcium Unsaturated

lipids or neutral hydrophobic
buffered formalin lipids– pink
Free fatty acids –
pink to blue
Phospholipids --
blue

Acetone Nile blue phospholipids Formal calcium

sulfate or neutral Phospholipids –
buffered formalin blue

Fluorescence microscopy: - Many lipid substances fluoresce in

ultraviolet light (oxidation products of cholesterol and of various
unsaturated fatty acids, vitamin A, etc) but only the fleeting green
 fluorescence of vitamin A is characteristic enough to be useful in
Histochemistry.

• Emission of light by a material when stimulated by absorption of

radiation.
Primary fluorescence; exhibited by the oxidation products of lipid
known as lipofuscins.
Secondary fluorescence; exhibited by fluorochromes dissolved in
lipids.

UV METHOD FOR LIPOFUSCINS

Fixation
• Cryostat sections (5-10 μm) air-dried; frozen sections of fixed
tissue; paraffin sections
Method
• Bring sections to xylene and mount in DPX
• Examine by fluorescence microscopy (dark ground transmission)
or epi-illumination
Results
• Lipofuscin ---- orange-yellow fluorescence
Lipofuscin In Neurons Of Human Brain Visualized Under UV Light
(Orange-yellow Fluorescence).Photo credit: Guardo, 2015

SECONDARY FLUORESENCE METHOD

Fixation and sections
Formalin or formal-calcium fixed frozen section
Procedure
1. Wash sections in distilled water
2. Stain for 3 mins in 0.1% aqueous phosphine 3R
3. Rinse quickly in distilled water and mount in 90% glycerin.
4. Examine by fluorescence microscopy
Results
Simple and compound lipids … silvery white fluorescence
Derived lipids……negative
A. Polarization microscopy:- Polarized light microscopy is a classic
and still useful technique for studying the ultrastructure of different
biological structures. The potential of polarized light microscopy is
greatly enhanced by combining staining and histochemical
reactions (Schlammadinger et al, 2005).
The birefrigent lipids found in mammalian tissues are
usually free cholesterol or its crystalline esters (Adams & Bayliss
1974). Polarization microscopy used to be considered a valuable
means for the distinction of doubly refractile cholesterol from other
lipids.

It has also been used to distinguish between glycerol esters and

cholesteryl esters since both groups stain identically in Sudan dyes
whereas the cholestryl esters unless in crystalline form display a
distinctive type of double refraction in polarized light (Maltese
cross birefringence). This is due to the liquid crystalline structure
of esters and this physical state is usually due to the admixtures of
phospholipids and is influenced by temperature, fixation and the
presence of water. `
However, Lison 1933 showed that its results could not be
interpreted in a chemically meaningful way. In the case of lipids,
birefringence appears to depend largely on factors other than
chemical constitution (such as state of aggregation, super cooling,
the nature of the mounting medium, etc.) even in the case of pure
compounds; the behavior of mixtures is unpredictable. Also the
melting points of lipids determine its appearance in polarized light.
The sensitivity of the method is poor because lipid mixtures
containing less than 5 per cent cholesterol show no birefringence
and even a positive result is not necessarily evidence of the
 presence of cholesterol (Gomori, 1952) and also certain non-lipid
structures like amyloid and glove powder can also appear
birefrigent.

POLARIZATION MICROSCOPY PROCEDURE

Unstained, formalin or formal-calcium fixed frozen sections are mounted

with an aqueous mountant and examined with the polarizing microscope.
Sections may be warmed at 37ºC for a few moments before re-
examination

CONCLUSION
• Lipids are fats or fat-like substances capable of being metabolized
by animals and vegetative organisms.

• Differences in physical properties of lipids determine their

behavior during staining.

• Specific chemicals are used for the demonstration of most lipid

classes.

• Lipid Histochemistry can be applied in certain disease processes.

REFERENCES
Baker, J. R. (1944) Quarterly Journal of Microscopical Science., 85.
Bancroft-J.D and M. Gamble (2008) Theories and Practice of Histolgical
Techniques. Churchill Living Stone, 6th Edition.
Cain, A. J. (1947) use of nile blue in examination of lipoids. Quarterly
Journal of microscopical Science., 88; 383.
Cain, A. J. (1949b) A Critique of the Plasmal reaction, with remarks on
recently proposed techniques. Quarterly Journal of Microscopical
Science., 90:75
Dunningan M.G. (1968) The use of Nile Blue Sulfate in the
histochemical Identification of phospholipids. Stain technology
43:249
Elleder M., Lodja Z. (1973) New, Rapid, Simple and selective method for
the demonstration of phospholipids. Histochemie. 28:68
Gomori, G. (1952) Microscopic Histochemistry, Chicago.
Keilig, I. (1944) Virchows Arch. Path. Anat., 312, 405
Lison, L., and Dagnelie, J. (1935) Bull. Histol. Appl. Phy
Zuhal A. (2005) Effect of High fat diet induced Obesity on female rat
liver. A histochemical study. European Journal of General
Medicine: 100-109