NEUROHISTOCHEMISTRY II

DEMONSTRATION
TECHNIQUES
BY
DR. MAHMOOD SEIDU
 TECHNIQUES FOR
DEMONSTRATING NEURONS
• There is an extensive range of histochemical techniques available for
investigating neuropathological complains.

• This is because nerve cells are generally larger than ordinary cells and has a
complex morphology. (elongated axons and complex interconnections with
one another or with organs).

• There are techniques for demonstrating the general architecture of nerve

cells; others are designed for specific cytoplasmic elements as well as axons
and dendrites.
DEMONSTRATION OF NEURON
ARCHITECTURE
• Hematoxylin and Eosin

• Hematoxylin van Gieson

• Fluorescent injection

• Formol-Thionin block impregnation

Hematoxylin and eosin & Hematoxylin van
Giessen methods
• The general architecture of neurons can conveniently be
seen in routine H & E preparations.

• However, the haematoxylin van Giessen technique is much

more popular than the H & E method.

• This is because the van Giessen stain highlights vascular

changes and emphasizes myelin staining as well as crisps
cellular cytology.
HAEMATOXYIN & EOSIN
HAEMATOXYIN Van GIESON
 Fluorescent injection method
• The morphology of individual neurons can be
studied by intracellular injection of a fluorescent
dye in fixed brain slices.

• This technique uses micromanipulators to

localize the micropipettes so that the dye can be
delivered into a single nerve cell
Fluorescent injection method
 Formol-Thionin block impregnation 1-2
• This technique was originally introduced by Golgi.

• In the original technique, fresh tissue was hardened with

potassium dichromate followed by impregnation in weak
silver nitrate solution.

• This method has gone through several modifications to the

current method of hardening with formalin containing
fixatives followed by thionin.
 Formol-Thionin block impregnation 2-2
• The method gives a tremendous insight into the
three dimensional nature of the neuron and its
processes.

• An unexplained phenomenon which makes the

method so useful is that only a few cells are
impregnated, giving a clear picture of neuronal
architecture undisturbed by surrounding
processes.
 DEMONSTRATION OF NERVE FIBRES
• There are several methods of demonstrating special
structures of the neuron.
• These structures include:
• axons,
• dendrites,
• terminal boutons (synaptic structures)
• Degenerated axons.
• Most of these methods are silver impregnation
techniques.
 SILVER IMPREGNATION
TECHNIQUES 2/2
• The technical explanation of silver impregnation has never been
well explained but when viewed from the simple understanding
of basic applied sciences one explanation can be accepted.

• During the process of impregnation silver foci are selectively

formed on targeted tissue components.

• With more silver ions and the presence of reducing agents

available, each silver focus appears to function as a macroscopic
aggregate of silver micronuclei catalyzing the reduction and
further precipitation of silver on and around it.
 IMPLICATION OF THE PRINCIPLE
• The implication is that factors that affect the redox potential
of reactants will affect silver impregnation techniques and
these can affect the staining procedure. The factors are:
• pH,
• temperature,
• concentration of reactants
• presence of interfering chemical ingredients to
redox activities
Silver impregnation methods therefore have three areas of
concern:

1.Reactants that induces the selectivity.

2.Those that cause the reduction

3. Those that intensify the results

 DEMONSTRATION OF AXONS &
PROCESSES
For axons and neuronal processes in the CNS there are two
important stains. Both of which are applicable on paraffin processed
sections.

• Bielschowsky

• Marshland, Glees & Erikson methods.

• Axons in peripheral nerves can be demonstrated by Palmgreen

method and Linder’s method all on paraffin sections.
 Bielschowsky silver Method
• Bielschowsky's silver stain is a very useful tool to detect
nerve fibers.

• It can be used to stain axons, neurofibrils and senile

plaques in the central nervous system.

• This method is easy to perform and is routinely used in the

study of Alzheimer's disease
1. Deparaffinize and hydrate sections to dH2O. Wash 3X for 3 min. in dH2O.

2. Place sections in 50 ml 10% silver nitrate in dark at 37ºC for 30 min. Keep this solution after incubation (for use in step 4).

3. Wash 3X for 3 min in dH2O.

4. Add concentrated ammonium hydroxide dropwise with stirring to the silver nitrate solution reserved from step 2. Add only
enough to dissolve the dark initial precipitate but not more.

5. Incubate sections in this solution for 15 min at 37ºC. Again, save this solution for use in step 6.

6. Wash sections in 0.1% ammonium hydroxide 3X for 2 min at room temperature.

7. Add 350 μl developer solution (0.2 ml 37% formaldehyde, 12 ml dH2O, 12.5 μl 20% nitric acid and 0.05 g citric acid) to the
silver hydroxide solution saved from step 4.

8. Stain sections in this solution for 10 min until they turn black.

9. Wash in 0.1% ammonium hydroxide 3X for 2 min and dH2O 3X for 2 min.

10. Tone in 0.2% gold chloride for 5 min.

11. Fix in 5% sodium thiosulfate for 1 min.

12. Wash in dH2O, dehydrate in alcohols then xylene and mount.
Axons stained using Bielshchowsky's silver
stain. 8 µm thick paraffin embedded sections
of mouse cerebellum
IMMUNOHISTTOCHEMICAL MARKERS FOR
NEURONS
The main markers can be divided into three groups
• Neuronal cytoskeletal proteins.

• Neuro-specific cytoplasmic proteins

• Protein associated with neuro-secretory granules

 Neuronal cytoskeletal proteins
• These are in the form of (neurofilament proteins), a form of
intermediate filament specific to nerve cells.

• Antibodies to neurofilament proteins identify mature nerve

cells and there are three types: Each have different molecular
weight.

• NF70 (L),
• NF150 (M)
• NF200 (H)
 Neuron-specific cytoplasmic
proteins
• Protein Gene Product (PGP) 9.5 and neuron specific
enolase (NSE) are proteins which are expressed at high
levels in neurons and can be reliably detected by
commercially available antibodies.

• They are however not specific for neuronal tissues and

should be used among a panel of antisera for reliable
identification.
Protein associated with neuro-
secretory granules
• These are used for establishing neuroendocrine
differentiation. Antibodies to these proteins will help stain
sites of synaptic junctions and normal cerebellar cortex.

• Chromogranin A is a protein of the dense core matrix of

neurosecretory granule.

• Synaptophysin is a membrane glycoprotein seen in pre-

synaptic neuro-secretory granules.
 DEMONSTRATION OF NORMAL
MYELIN
• Myelin is formed by oligodendrocytes in the CNS and Schwann cells in
the PNS.

• Each nerve is wrapped up in up to 100 concentric layers of highly

specialized plasma membrane.

• Chemically, myelin consists of specific proteins, lipids and cerebrosides.

• Its demonstration can be achieved by using immunohistochemical

techniques or conventional methods.
Immunohistochemical methods of detecting normal myelin

• The presence of myelin can be demonstrated by

immunohistochemistry with S100 protein and Leu-7
antibodies.
Other myelin marker antibodies are
• Galacto-cerebroside antibodies
• Myelin basic protein (MBP)
• Myelin associated glycoprotein (MAG)
• Carbonic anhydrase –C
• Alpha/Beta crystalline.
Immunohistochemical methods of detecting normal myelin

• None of these markers can replace conventional

stains in the demonstration of normal myelin.

• S100 has been very useful in detecting tumours

derived from cells forming myelin in peripheral
nerves (Schwann cells).
IHC for MYELIN & HX COUNTER STAIN
Conventional methods of detecting
normal myelin
• The conventional methods of staining myelin
rely on the detection of the components of the
Schwann cell membrane.

• The main detectable components are:

• sphingomyelin and cerebrosides.
 Sphingomyelin:
• Sphingomyelin is a phospholipid found in abundance in
Schwann cell plasma membrane Therefore lipid
histochemical stains can be used for this purpose.

• Myelin has a strong affinity for haematoxylin after

treatment with chromate-containing fixative, methods
in which metallic salts are used to mordant myelin
dyes has been useful.
 Sphingomyelin:
• The dichromate-acid haematin (DAH) method is
reasonably specific for choline containing
phospholipids.

• Interference from proteins, which are also positive for

this stain can be discounted by including a lipid
extracted control section
 Cerebrosides
• These lipids can be demonstrated by reactions
specific for their carbohydrate molecules.

• Cerebrosides and related lipids are PAS

positive.

• In this process periodic acid converts 1:2 glycol

groups to Schiff’s stainable aldehydes.
DEMONSTRATION OF DEGENERATION PRODUCTS OF
MYELIN
• In de-myelinating diseases or following neuronal death, the axon and
associated myelin dies.

• Myelin loss will occur in such processes as:

• infarction,
• multiple sclerosis
• and several primary degenerative disease of the central nervous
system.

• In such circumstances the detection of degeneration products is

important in neuropathology.
DEMONSTRATION OF DEGENERATION PRODUCTS OF MYELIN

• In its normal state myelin is hydrophilic due to its high

content of polar phospholipids but following
degeneration, it becomes hydrophobic with the
formation of cholesterol esters.

• Both normal myelin and degenerated myelin can be

stained by osmium tetraoxide.
DEMONSTRATION OF DEGENERATION PRODUCTS OF MYELIN

• The osmiumphilia of the normal myelin can be

blocked by pretreatment with strong oxidizing agents
(potassium dichromate) and this phenomenon allows
the differentiation of degenerate myelin from normal
ones.

• (this is the principle of Marchi method for degenerate

myelin).
 LOSS OF NORMAL MYELIN
STAINING
• Following myelin loss, the degenerate myelin is
phagocytized my macrophages and over a period
of weeks or months they are removed from the
area.

• This process results in the lack of normal and

degenerated myelin staining with routine
methods
 COMBINATION TECHNIQUES FOR DEGENERATE
MYELIN
• Stains for neutral lipids such as oil red O can effectively
demonstrate myelin degeneration in formalin-fixed frozen sections
during the period of myelin-lipid phagocytosis.

• This is an effective way for looking for tract degeneration when loss
of normal myelin staining is established but no Marchi method is
available.

• Oil red O can be combined with dichromate acid hematein (DAH)

to stain normal myelin blue to contrast beautifully with the red
stained fatty degenerate myelin lipids within lipophages.
 DEMONSTRATION OF NISSL GRANULES
• Nissl stains are used routinely in neurohistochemistry not
only to demonstrate nissl granules but also to demonstrate
the cellular pattern.

• The granules are distributed only in the cytoplasm of nerve

cells and dendrites.

• Nissl granules can be demonstrated by several basic dyes.

These include: Neutral red, methylene blue, pyronin, Azur,
thionin, toluidine blue and cresyl fast violet.
 DEMONSTRATION OF NISSL
GRANULES
• Tissues fixed in alcohol stains particularly well in toluidine blue
and cresyl fast violet. Cresyl fast violet is however the method
of choice for Nissl substances.

Nissl substances ……………………purple dark blue, Neurons

……………………………….. pale purple blue nuclei
…………………………………….purple blue.

• When the emphasis of demonstration is on Nissl granules, the

stain should be acidified with the addition of 0.25% acetic acid
 MELANIN
• Melanin is produced from tyrosine through the action of an enzymes,
tyrosinase (DOPA-oxidase).

• The enzyme acts slowly on tyrosine to produce DOPA (dihydroxy-

phenylalanine) and then rapidly to an intermediate pigment by the same
enzyme.

• The intermediate pigment then polymerizes to melanin which are bound to

protein and localized in the cytoplasm as melanin granules (melanosomes).
 MELANIN IN THE BRAIN
• Melanin is found particularly in the substancia nigra of
the brain

• It is found in such quantities that they can be observed

macroscopically as a black streak in this area.

• In patients with long standing Parkinson’s disease, this

streak is markedly reduced.
 DEMONSTRATION OF MELANIN
Methods used to identify melanin and melanin producing cells.
• Fluorescent methods

• Immunocytochemistry

• Enzyme methods

• Solubility and bleaching methods

• Reducing methods
 DEMO. OF MELANIN:
Fluorescence
• Fluorescent methods (formalin induced fluorescence)
• Certain aromatic amines including DOPA and Dopamine
show yellow primary fluorescence when they are
exposed to formaldehyde.

• Any melanin precursors present in tissues will form a

product of isocarboline derivative which show a yellow
fluorescence.
 DEMO. OF MELANIN :Immuno &
enzyme
• IMMUNO: There’re few antibodies that can be used to
demonstrate melanocytic lesions. The most widely used
antibodies are. S100, HMB-45 and PGP 9.5.

• ENZYME (DOPA): Cells that are capable of producing

melanin can be demonstrated.
• The enzyme tyrosinase within these cells is activated to
oxidize DOPA to form an insoluble brown-black pigment
 DEMO. OF MELANIN :Solubility &
bleaching
• Solubility and bleaching
• Melanins are insoluble in most organic solvents. It will therefore require a
strong oxidizing agent to bleach melanin and the blacker the melanin the
longer it takes to bleach

• Among the many strong oxidizing agents available, per acetic acid is the
oxidant of choice.
• However treatment with potassium permanganate followed by oxalic acid
has worked well with some workers.

• Other oxidants are, chlorate, chromic acid, and peroxide

 Demo of Melanin: reducing
methods
• Melanin is a powerful reducing agent and this property is used in
demonstrating its presence in two different methods.

• Masson-Fontana method: Ammoniacal silver solution is reduced

to metallic silver without the use of external reducer. (the
argentaffin properties of melanin).

• Melanin also has argyrophilic properties where it can be

blackened by silver impregnation methods by employing external
reducers. (this method is of no diagnostic significance).
 LIPOFUSCIN
• These are yellow-brown to reddish-brown pigments
occurring widely throughout the body.

• They are thought to be produced by the oxidation process

of lipids and lipoproteins.

• Lipofuscin pigment, often called "wear-and-tear" pigment,

is easily seen in nervous tissue and in areas of high
macrophage activity.
 LIPOFUSCIN
• The oxidation process occurs slowly and
progressively so that the pigments exhibit variable
staining reactions leading to different colours, varied
shapes and sizes.

• In many occasions they are found in association with

other pigments.
 Demonstration of Lipofuscin
• Because lipofuscin is formed by a slow progressive
oxidation of lipid and lipoproteins, histochemical
reactions will vary according to the degree of oxidation.

• It is advisable to perform a variety of techniques in other

to be sure that the pigment is lipofuscin.

• This is because almost all the methods used in the

demonstration of lipofuscins are designed for other
pigment as well.
 Demo. of Lipofuscin
• The most common and useful methods are:
• ►PAS
• ►Schmorl’s ferric ferrocyanide
• ►Long ZN staining
• ►Sudan Black
• ►Gomori’s aldehydes fuchsin
• ►Masson-Fontana silver method
• ►Methyl green stain
 DEMONSTRATION OF EPENDYMAL CELLS
• Ependymal cells are ciliated and are not lined by basement
membranes. These can be demonstrated easily by
conventional H and E staining.

• Ependymal cells may form tumours of the central nervous

system where their identification is based on the
determination of cilia basal body (blepharoplast).

• This can be done conveniently by PTAH or iron hematoxylin.

 DEMONSTRATION OF EPENDYMAL CELLS
• Immunohistochemically, ependymal cells may
express the glial intermediate filament (GFAP)
but do not express the normal epithelial
intermediate filament (cytokeratin)
 DEMONSTRATION OF ASTROCYTES 1/4
• The identification of astrocytes is important in
neuropathology as they react promptly to local tissue injury.

• They increase in size in response to cerebral edema or to

metabolic disturbances such as liver failure.

• In such circumstances they characteristically develop

prominent pink cytoplasm, the nucleus become eccentric
and the processes prominent.
 DEMONSTRATION OF ASTROCYTES 2/4
• Such cells are referred to as reactive astrocytes.

• Astrocytes proliferate when there is damage to the

CNS and fill defects left by loss of specialized nervous
tissue with glial fibres.

• This is called astrocytic gliosis and its identification is

necessary for the analysis of disease of the CNS
ASTROCYTIC GLIOSIS (PROLIFERATION OF ASTROCYTES
 Demonstration of Astrocytes 3/4
• Astrocytes are star-shaped but contrary to common sense
the processes are not easily displayed in H & E sections.

• Using Cajal’s gold sublimate method, two types of

astrocytes can be identified (fibrous and protoplasmic
types).

• Fibrous astrocytes are found mainly in the white matter.

They have small cell bodies with long processes.
 Demonstration of Astrocytes 4/4
• Protoplasmic astrocytes have more processes
that are shorter and thicker than those of the
fibrous type.

• Immunohistochemical demonstration of
astrocytes is mainly by the expression of glial
fibrillary acidic protein (GFAP)
GFAP IHC POSITIVE FOR ASTROCYTES
 DEMONSTRATION OF OLIGODENDROCYTES 1/2
• In H & E, oligodendrocytes are identified by their small dense
rounded nuclei and cytoplasm not distinguishable from the
surrounding tissues.

• In formalin-fixed paraffin processed sections the cytoplasm

may form an artefactual halo around the nucleus.

• Demonstration of oligodendrocytes is merely an anatomical

or histological exercise. It is called for in fresh human tissue,
in autopsy material, autolytic processes frequently lead to
poor results.
 DEMONSTRATION OF
OLIGODENDROCYTES
• Immunohistochemical markers for oligodendrocytes
are antibodies to galactocerebroside, myelin basic
protein or carbonic anhydrase-C.

• Tumours derived from oligodendrocytes do not

express these, given rise to speculations that
oligodendroglioma may not actually be a tumour
originating from oligodendrocytes.
OLIGODENDROCYTES SHOWING PERI-NUCLEAR HALO
 DEMONSTRATION OF MICROGLIA 1/2

• Microglia are cells of the mononuclear phagocytic

system.

• Inactive microglia are static cells in the normal CNS

with dendritic morphology called resting microglia.

• Following brain injury these inactive microglia undergo

phenotypic changes by expressing cell surface markers
more like peripheral macrophages and become
phagocytic.
 DEMONSTRATION OF MICROGLIA 1/2

• In conventional stained sections microglia cells

appear as rod-shaped nuclei.

• In silver stained sections, fixed microglia may be

seen to have numerous branching processes.
 DEMONSTRATION OF MICROGLIA
2/2
• Immunohistochemistry is the most reliable method for demonstrating microglia.
• They stain positively to antisera which detect macrophages:

• CD 68 – a peripheral macrophage marker

• EMB/11 works in frozen sections only

• KP-1: HLA-DR a class II MHC protein

• CD 45 is faintly positive
microglia cells appear as rod-shaped nuclei
fixed microglia may be seen to have numerous branching processes.
The End