LIPIDS

LIPIDS
 Any group of fats or fat-like substances characterized by
their insolubility in water. These fats would include:
 True fats- ester of fatty acid and glycerol
 Lipids- phospholipids, cerbrosides and waxes
 Hydrocarbons- squalene and carotene

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CLASSIFICATION

 Simple lipids- usually found in the body as energy stores in

adipose tissue
 Waxes are usually found in plant and animal species
 Compound lipids can be formed in the brain and central
nervous system
 Derived lipids can originate from the simple and
compound lipids by means of hydrolysis
 Examples: cholesterol, bile acids and sex and adrenocortical
hormones
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CLASSIFICATION

 Lipids can be demonstrated by standard procedures, but

exposure to alcohols, acetone, chloroform, xylene and paraffin
will destroy them
 Frozen sections- best to demonstrate simple lipids
 phospholipids, lipofuscin and granules of leukocytes can bind to
other tissue elements or entities and resistant to paraffin
processing
 Formal calcium-fixative of choice for phospholipids (also
demonstrated using oxidation and mordanting solutions or
Sudan dyes
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CLASSIFICATION- nonpolar lipids
Features Iinfluencing
Class Member
Histochemistry
Melting point and sudanophilia depends on
Fatty acids
1. number of double bonds.. HYDROHPOBIC
Unconjugated Melting point 144 C, therefore not
lipids Cholesterol sudanophilic at RT. Birefringent in polarized
light. HYDROPHOBIC
Cholesterol esters, Melting point depends upon degree of
2. Esters mono-, di-, and saturation of constituent fatty acids.
triglycerides HYDROPHOBIC

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CLASSIFICATION- polar lipids
Features Iinfluencing
Class Member
Histochemistry
1. Phospholipids Phospholipids contain phosphoric acid, long-chain
F.A., polyhydric alcohols, and variable nitrogenous
Glycerol-based bases. HYDROPHILIC
Phosphatidyl F.A. linked to glycerol with an ester bond which is
choline (lecithins) alk. Labile. Contain choline. BASIC
Phosphatidyl serine Ester bond. ACIDIC
Phosphatidyl Ester bond. BASIC
ethanolamine
(cephalins
Plasmalogens Ester and other bonds. BASIC

Sphingosine-based Sphingomyelins One sat. F.A. linked to sphingosine by an alk-

resistant amide bond plus phosphoryl choline.
BASIC

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CLASSIFICATION- polar lipids (conti…)
Features Iinfluencing
Class Member
Histochemistry
2. Glycerol- Cerobrosides Cerebrpsides contain a hexose linked to
based a ceramide, usually glucose. NEUTRAL

Sulfatides Sulfate esters of cerobrosides, HIGHLY

ACIDIC.

Gangliosides Contain N-acetyl neuramic acid (sialic

acid). Water-soluble unless complexed
with protein and/or phospholipids. acidic

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FIXATION
 Frozen section- most common method of demonstrating
lipids
 Osmium tetroxide and chromic acid- the only reagents
that truly fix lipids, however, cause great alteration in the
chemical reactivity of lipids
 Formal calcium-choice of fixative for lipid histochemistry
and is prepared by adding 2% calcium acetate to 10%
formalin
 Formal saline should be avoided when dealing with any
material which may require examination of lipids
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MICROTOMY

 Frozen or cryostat sections require for lipid histochemistry

 Have morphological appearance that is at least as good
as that of a routine paraffin section
 Mounted directly on slides
 Formal-calcium-fixed blocks of tissue are also suitable for
cryostat sectioning but is advisable to mount sections in
chrome-gelatin- coated slides or used of ‘charged’ slides

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HISTOPHYSICAL METHODS
•Optical properties
•Affinity to fat stains and Sudan dyes

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HISTOPHYSICAL METHODS
 Microscopic discrimination between classes of lipids
based on their differences in physical property
 Optical properties
 Affinity to fat stains and Sudan dyes
 Example between crystalline and liquid lipids and
hydrophobic and hydrophilic types

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Birefringence of Lipids

 Discrimination between fatty droplets that are stained

and are non-birefringent (isotropic) from crystalline lipids
which remain unstained but appear anisotropic
(birefringent)
 Polarizing microscopy also distinguish cholesterol ester
from glycerol esters, where cholesteryl ester shows
‘Maltese cross’ birefringence

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Fat Stains & the Sudan Dyes
 Sudan III- first Sudan dye introduced into histochemistry
 Sudan IV- preferred by Michaelis (1901) because of its
darker staining
 Oil red O- more intense in staining and generally more
preferred to the other red dyes
 Sudan Black B – most sensitive and versatile and was
introduced by Lison and Dagnelie (1935)

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Fat Stains & the Sudan Dyes
 Oily and greasy hydrophobic lipids, have an affinity for the
Sudan dyes
 Staining is based on the fact that dyes are more soluble
in tissue fats than in their dye solvent and by adsorption
process
 Sudan III- first Sudan dye introduced into histochemistry
 Sudan IV- preferred by Michaelis (1901) because of its
darker staining

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Fat Stains & the Sudan Dyes

 Organic solvents used to facilitate ease of

penetration of Sudans to fats
 Isopropanol‘
 Triethyl phosphate
 Propylene glycol
 70% ethanol for Oil red O and Sudan black

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Oil Red O in dextrin

 Fixation: fresh frozen or NBF, rinse, frozen

 Sections: 5 um mount on SuperFrost/ plus slides, air dry
 Method:
 place slides directly into filtered 0.5% Oil red O in dextrin.
Stain for 20 minutes, rinse with running water briefly.
 Counterstain with Gill II hematoxylin for 20-30 seconds.
Rinse with water, blue , cover slip with aq. Mounting
medium
 Results: Fat- brilliant red, nuclei- blue
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Standard Sudan black method for
fats and phospholipids
 Fixation and Sections: cryostat sections post-fixed in
formal calcium; short fixed frozen sections; unfixed
cryostat sections (preferred)
 Results:
 unsat. esters- & TAG- blue-black
 phospholipids-gray
 myelin- bronze dichroism in polarized light

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Bromine Sudan Black method for
Lipids
 Fixation and sections: cryostat sections post-
fixed for 1 hour in formal calcium; short-fixed
frozen sections
 Results:
 phospholipids-gray

 sphingomyelin- bronze in polarized light

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Nile Blue Sulfate method for acidic
& neutral lipids
 Comprises two components
 Red oxazone- dissolves in neutral lipids
 Blue oxazine- reacts with phospholipids and free F.A.
 pH 9.0 and interference by non-lipid structures is reduced
by employing an acid-hydrolyzed dye solution

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Nile Blue Sulfate method for acidic
& neutral lipids
 Fixation and sections: cryostat sections post-fixed for 1
hour in formal calcium; short-fixed frozen sections
 Results:
 unsat. Hydrophobic lipids- pink
 Free fatty acids- pink to blue
 Phospholipids- blue

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Acetone- Nile blue sulfate method
for phospholipids
 A Dunnigan’s modification whereby fatty acids are first
extracted with acetone so the Nile blue sulfate staining
will subsequently be confined to phospholipids
 Fixation and sections: cryostat sections post-fixed for 1
hour in formal calcium (converted into a soap before
fixation); short-fixed frozen sections
 Results: phospholipids- blue

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HISTOCHEMICAL METHODS
•Include methods for each lipid class and other relevant
techniques

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HISTOCHEMICAL METHODS for
Free Fatty Acids
 Weigert’s lithium hematoxylin- developed by Fischler
(1904) to stain copper soaps bound with free F.A.
 based on the observation that free fatty acids bind heavy metal
ions to form soaps
 Okamoto et. Al. procedure- use
dimethylaminobenzylidine rhodanine for the same purpose
 Neither method is specific as Holczinger’s copper
rubeanic acid method for free F.A.

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Copper Rubeanic Acid method for
free fatty acids
 Visualized copper soaps with rubeanic acid, using EDTA to
remove extraneous copper
 Fixation: Cryosat sections post-fixed in formal calcium;
fixed frozen sections
 Results:
 Free F.A.- dark green (rubeanic acid) or red (p-
dimethylaminobenzylidine rhodanine
 This is absent from acetone-extracted control
 If cryostat sections are used, extraction is best performed
before post-fixation
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Holczinger’s Copper Acetate
procedure
 Provides an equally specific and sensitive alternative using
Okamoto’s rhodanine reagent with rather less background
staining and is compatible with hematoxylin nuclear stain
 Calcium and iron deposits can be verified by extraction
with 1% HCl (for calcium) or 5% oxalic acid (for iron salts)

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HISTOCHEMICAL METHODS for
Cholesterol
 Schultz (1924) technique- adapted the Liebermann- Burchardt
reaction to demonstrate cholesterol in tissue sections
 Method: oxidation in air with iron alum followed by treatment with a
sulfuric-acetic acid mixture
 Results: free and esterified cholesterol – blue
 Roussouw et al- overcame technical problems associated with
Schultz procedure by a using a ‘pre-mixed reagent which is a
combination of ferric chloride and acetic, sulfuric, and
phosphoric acids
 Iodine-sulfuric acid technique- described by Okamoto but color
is transient and masked by iodine
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Adams’ Perchloric Acid-
naphthoquinone (PAN) method
 Depends on the different principle for cholesterol detection
 Sensitive, accurate and has more satisfactory tissue
preservation
 Ineffective unless cholesterol has been oxidized with ferric salts
or exposure to air (same is true with the procedures mentioned
beforehand)
 Fixation and Sections: Formal calcium fixed frozen section;
cryostat sections post-fixed in formal calcium
 Results: cholesterol and related steroids- blue

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Filipin Method for free
cholesterol
 Uses filipin, a compound similar to digitonin
 Used in aq. Media to complex with free cholesterol to
produce a highly fluorescent compound
 Used to detect accumulation of free cholesterolin
cultured fibroblasts from Niemann-Pick disease
 Fixation and section: cryosat section, post-fixed; frozen
sections of fixed tissue
 Results: free cholesterol –shows strong silvery
fluorescence
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Enzymatic methods for free
cholesterol
 Uses enzymes like cholesterol esterase and oxidase for
cholesterol hydrolysis and oxidation
 Practical value is limited by their cost and complexity
for routine light microscopy
 Proved to be useful at the EM level

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HISTOCHEMICAL METHODS for
unsaturated lipids
 UNSATURATED- SCHIFF METHOD -
 Fixation and sections: preferably unfixed cryostat sections
 Results: unsaturated lipids- magenta
 OSMIUM TETROXIDE METHOD –
 Fixation and sections: preferably unfixed cryostat sections
 Results: unsaturated lipids- brown to black
 Note: osmium solution must e handled inside a fume hood to
avoid effect of its toxic vapor o cornea and mucous membrane

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HISTOCHEMICAL METHODS for
triglycerides
 CALCIUM LIPASE METHOD
 Adams( 1966) use pancreatic lipase to hydrolyz triglycerides into
their constituents fatty acids
 Highly specific for trigylcerides and has been adapted for electron
histochemistry
 Uses cryostat sections post-fixed in formal calcium
 Results: triglycerides- brown

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HISTOCHEMICAL METHODS for
Glycerol-based phospholipids
 GOLD HYDROXAMIC ACID METHOD
 Recommended for selective demonstration of
phosphoglycerides (lecithin & cephalins)
 Ester bonds, converted to hydroxamic acids by alk.
hydroxamine, reduce silver nitrate to metallic silver
and finally stabilized by gold toning
 Phosphoglycerides are stained purple

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HISTOCHEMICAL METHODS for
Sphingomyelins
 DICHROMATE-ACID HEMATEIN (DAH) for choline-
containing phospholipids
 Unfixed cryostat sections are recommended
 Lecithin and sphingomyelin- blue black
 FERRIC HEMATOXYLIN METHOD
 Simpler, quicker, and more sensitive than DAH technique
 Method of choice for sphingomyelin
 Ideally unfixed cryostat sections are used
 Phospolipids and nuclei- blue
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HISTOCHEMICAL METHODS for
Sphingomyelins
 NAOH- FERRIC HEMATOXYLIN/DAH METHOD
 Ideally unfixed cryostat sections mounted on chrome-
gelatin-coated slides are used
 Sphingomyelin-blue
 Notes: to prevent interference by plasmlogens, sections are
treated with 1% mercuric chloride in 1% HCl for 10 minutes

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HISTOCHEMICAL METHODS for
other lipids
 Toluidine blue-acetone method for sulfatide
 Cryostat sections post-fixed with formal calcium; fixed
frozen sections
 Sulfatide deposits- metachromatic red-brown or yellow
 Modified PAS reaction cerobroside
 Cryostat sections post-fixed with formal calcium; fixed
frozen sections
 Cerebroside- magenta

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HISTOCHEMICAL METHODS for other
lipids
 Borohydride-periodate-Schiff (BHPS) method for
gangliosides
 Cryostat sections post-fixed with formal calcium; fixed frozen
sections
 gangliosides- red; nuclei- blue
 UV method for lipofuscin
 Cryostat sections (5-10 um) air dried; fixed frozen sections;
paraffin sections
 lipofuscin- orange-yellow fluorescence

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APPLICATION OF LIPID
HISTOCHEMISTRY IN PATHOLOGY

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MYELIN & DEMYELINATION
 Primary demyelination and dysmyelination are
demonstrated using paraffin sections
 Frozen section- preferred for demonstration of active
demyelination and stain with Sudan dye
 Lipophage- convenient hallmark of ongoing demyelination
 Dichromate-acid hematein- Oil red O combination-
recommended for demonstration of normal and degenerating
myelin
 Good color contrast between blue myelin and red-stained
esters within lipophages
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METHODS FOR DEMONSTRATION OF
normal and degenerating myelin
 Dichromate-acid hematein- Oil red O combination- recommended
for demonstration of normal and degenerating myelin
 Good color contrast between blue myelin and red-stained esters
within lipophages
 Sudan Black B- normal myelin- gray; degenerating myelin-intense
blue
 Luxol fast blue
 For paraffin sections
 Osmium tetroxide a-naphthylamine (OTAN) technique
 PAS Sudan red procedure of Hirsch & Peiffer
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METHODS FOR DEMONSTRATION OF
normal and degenerating myelin
 Parkinson’s disease and the characteristic Lewy bodies
can be demonstrated by:
 NAOH-ACID HEMATEIN PROCEDURE
 or SUDAN BLACK B- sphingomyelin is demonstrated with red
birefringence
 Sudan Black B is also use to demonstrate lipid deposition
in Fabry’s disease

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