STAINING OF CENTRAL NERVOUS

TISSUE
STAINING OF CENTRAL NERVOUS TISSUE

Central Nervous System

• Brain and Spinal Cord

Peripheral Nervous System

• Peripheral Nerves

Autonomic Nervous System

• Autonomic Nerves and Ganglia

THE NERVOUS SYSTEM
THE NERVOUS TISSUE

 The central nervous system is composed of neurons that

are supported by a framework of:

 Glial cells – Astrocytes, Oligodendrocytes and

ependymal cells

 Microglia

 The processes of these cells combine to form a delicate

fibrillary background termed neuropil.
THE NEURONS
 Neurons are morphologically
heterogenous, ranging from the
small round cells that populate
the internal granular layer of the
cerebellum, to the large pyramidal
Betz cells of the primary motor
cortex.

 The function of the neuron is to

receive and transmit electrical
signals.
ASTROCYTES

 Astrocytes represent the major supporting cells in the

brain.

 Astrocytes respond to injury by producing a dense network

of processes, somewhat analogous to the fibrous scar that
occurs elsewhere in the body.

 Astrocyte swelling is often associated with increase

synthesis of glial fibrillary protein, the astrocyte’s major
cytoskeletal protein.
OLIGODENDROCYTES

 Oligodendrocytes or oligodendroglial cells are recognize in

the routine sections by their small, rounded, lymphocyte-
like nuclei.

 Oligodendroglial cytoplasmic processes wrap around the

axons of neurons to form myelin, in a manner analogous
to Schwann cells of the peripheral nervous system.
STAINING OF CENTRAL NERVOUS TISSUE
Bielschowsky’s Technique
For Neurons, Axons and
Neurofibrils

Bodian’s Stain
For Nerve Fibers and
Nerve Endings

Sevier – Munger
Technique
 BIELSCHOWSKY’S
TECHNIQUE
FOR NEURONS, AXONS AND
NEUROFIBRILS

Reagents and
Its Preparation
• Staining Solution
(Ammoniacal silver)
• 20% Silver Nitrate – 5 mL
• 40 % Ammonium
hydroxide – 6 drops
• 28 % Ammonia Water – 5
mL or until the brown
precipitate is almost
dissolved
• Distilled water up to 25 mL
Notes and
Result Special
Consideration
• Neurofibrils, • Fixation: Formol Saline
axons,anddendrites – black
• Sections:
on a grayish background
• Paraffin Sections (7-10
• Neuroglia and collagen –
um)
lightly stained
• Frozen Sections (10-15
um)
 BODIAN’S STAIN
FOR NERVE FIBERS AND NERVE ENDINGS

Protargol
Solution
• Protargol – 1 gram
• Distilled water – 100 mL
• Pour distilled water into wide
mouth beaker set on plate at
37 degrees Celsius. Slowly
sprinkle the Protargol on the
surface of the water and
allow it to remain
undisturbed until it becomes
dissolved.
Hydroquinone
Solution
Aniline Blue Stain
(Reducing
Solution)
• Hydroquinone – 1 gram • Aniline blue – 0.1 gram
• Distilled water – 95 mL • Oxalic acid – 2 grams
• Formalin Reagent (37-40%) • Phosphomolybdic acid – 2
– 5 mL grams
• Distilled water – 300 mL
 BODIAN’S STAIN
FOR NERVE FIBERS AND NERVE
ENDINGS

Notes and
This stain is
Special
also used for:
Considerations
• Fixation: • Demonstration of neuritic
• Formol saline plaques

• Buffered Formalin • Demonstration of

• Sections: Paraffin section Neurofibrillary tangles

• Diagnosis of Alzheimer’s
diesease
 SEVIER – MUNGER TECHNIQUE

Sodium Ammoniacal
Staining
Carbonate Silver Solution
Solutions:
Solution (Working)
• 20% Silver nitrate • Sodium carbonate – 8 gm • 50 mL of 10% silver nitrate
solution, add 28%-30%
solution • Distilled water – 10 mL ammonium hydroxide until dark
• 10% Silver nitrate brown precipitate is disappeared.

solution • Shake vigorously between drops.

• Avoid complete decolorization.
• 5% Sodium thiosulfate
• Endpoint is slightly cloudy
solution solution
• Add to this 0.5 mL of sodium
carbonate solution. Shake well.
Solution is now crystal clear.
 SEVIER – MUNGER TECHNIQUE

• Axons – light brown

• Myelin sheath – black
• Neuritic plaques and tangles – black
Result • Argentaffin granules - black
• Large and small peripheral neuritis – black

•Fixation:
•10 % buffered formalin

Notes and •Avoid using chromate fixatives

Special •Sections:

Considerations
•Paraffin section cut in 6 um

•Sodium carbonate is in hydrated form

STAINING OF NISSL BODIES
Nissl Bodies

 coarse granules made up of

ribonucleic acid that are scattered in These substance are
stained by:
the cytoplasm of nerve cells, except
 Cresyl fast violet – for formalin
in the part from which the axon
fixed tissues
arises. Absence of which usually
suggests nerve cell degeneration.  Toluidine blue – for formalin fixed
tissues

 Methylene blue

 Thionine – for alcohol fixed tissues

 THIONINE STAINING
OF NISSL BODIES

Results:
Staining Solutions: Nissl granules and nucleoli:
Notes and Special
•0.5 % Thionine in 20 % •In Thionine: purple Considerations:
alcohol Phenol crystals •In Toluidine blue and Fixation:
•0.5 % Aqueous Thionine Polychrome methylene blue: Formol Saline
Acetic acid solution deep blue Buffered Formalin
Background – almost colorless
CRESYL FAST VIOLET (NISSL) STAIN
FOR PARAFFIN SECTIONS

Staining solutions:
Cresyl fast violet – 0.5 grams
Distilled water – 100 mL
Differentiation solutions
Glacial Acetic Acid – 250 uL
Alcohol – 100 mL

Notes and Special Considerations:

Result: Fixation:

Nissl Substance – purple dark blue Alcohol

Carnoy’s fluid
Neurons – pale purple blue
Neutral formol saline
Cell nuclei – purple blue
Sections: Paraffin Section (7-10 um)
STAINING FOR MYELIN
SHEATH
Weigert-Pal technique of Staining
Normal Myelin Sheaths

Kluver& Barrera Luxol Fast Blue Stain

for Myelin with Nissl Counterstain

Luxol Fast Blue- H & E Stain for Myelin

Luxol Fast Blue-PAS-Hematoxylin Stain

for Myelin

Weil’s Method for Myelin Sheaths

WEIGERT-PAL TECHNIQUE
OF STAINING NORMAL MYELIN SHEATHS

Staining solutions:
10% hematoxylin in absolute alcohol –
10 mL Decolorizer:
Distilled water – 9m mL 0.5 – 1 % iron alum
Saturated aqueous lithium carbonate –
1-2 mL

Differentiate:
0.25 % Potassium permanganate for 30 Counter Stain:
seconds to 3 minutes until white matter Neutral red (if desired)
is distinguishable from gray matter
WEIGERT-PAL TECHNIQUE
Result
OF STAINING NORMAL MYELIN SHEATHS

• Myelin – blue black

• Cells – brown

Notes and Special

Considerations
• Fixation:
• Orth’s fluid (48 hours)
• Transfer to 2.5 % Potassium dichromate
for 2 – 6 days
• Sections:
• Paraffin Sections(10-12 um)
• Celloidin is considered better
 KLUVER& BARRERA LUXOL FAST BLUE
STAIN
FOR MYELIN WITH NISSL COUNTERSTAIN

Luxol Fast Cresyl Violet Cresyl Violet

Blue Solution Solution Differentiator
• Luxol fast blue - 1 gm. • Cresyl violet - 0.5 gm. • Alcohol - 100 ml.
• Methanol (absolute) - • Distilled water - 100 ml. • Glacial acetic acid - 250
1000 ml. μl.
• 10% Acetic acid - 5 ml.
• Mix reagents and filter.
This may then be stored
for up to 18 months
before use.
KLUVER& BARRERA LUXOL FAST BLUE
STAIN
FOR MYELIN WITH NISSL COUNTERSTAIN

Result
• Myelin – blue or green
• Cells – violet or pink

Notes and Special

Considerations
• Fixation: Formalin
• Sections: Paraffin 10 to 15 μm
 LUXOL FAST BLUE- H & E STAIN
FOR MYELIN

Lithium Harris’ Alum

Luxol Fast Blue
Carbonate Hematoxylin
Solution
Solution Solution
• Luxol fast blue - 1 gm. • Lithium carbonate – • Acid alcohol
• Methanol (absolute) - 0.05 gm • 70% alcohol – 100 mL
1000 ml. • Distilled water – 100 mL • Hydrochloric acid – 1
• 10% Acetic acid - 5 ml. mL
• Mix reagents and filter.
This may then be stored
for up to 18 months
before use.
 LUXOL FAST BLUE- H & E STAIN
FOR MYELIN CONT: EOSIN-PHLOXINE SOLUTION

Harris’ Alum
Stock Eosin Stock Phloxine
Hematoxylin
Solution Solution
Solution
• Water soluble Eosin Y – • Phloxine B – 1 gm • Stock eosin – 100 mL
1 gm • Distilled water – 100 mL • Stochphloxine – 10 mL
• Distilled water – 100 mL • 95 % Alcohol – 780 mL
• Glacial acetic acid – 4
mL
LUXOL FAST BLUE- H & E STAIN
FOR MYELIN

Results:
Myelin – blue green
Nuclei – dark blue
Cytoplasm – various shades
of pink

Notes and Special Considerations:

Fixation: 10% Formalin
Sections:Paraffin sections (6
um)
 LUXOL FAST BLUE-PAS-HEMATOXYLIN
STAIN
FOR MYELIN
Lithium
Luxol Fast Blue
Carbonate Schiff’s Solution
Solution
Solution
• Luxol fast blue - 1 gm. • Lithium carbonate – • Bring to boil 200 mL of distilled
water and remove from flame.
• Methanol (absolute) - 0.05 gm
• When bubbling ceases, add 1g of
1000 ml. • Distilled water – 100 mL basic fuchsin and stir until
dissolved.
• 10% Acetic acid - 5 ml.
• Cool to 25℃.
• Mix reagents and filter.
• Add 1g of anhydrous sodium
This may then be stored bisulfite.
for up to 18 months • Keep in dark for 2 days at room

before use. temp.

• Store in brown bottle and
refrigerate
LUXOL FAST BLUE-PAS-HEMATOXYLIN
STAIN
FOR MYELIN

Sulphurous 0.5% Periodic

Acid Solution Acid Solution
• 10% aqueous Solution Sodium • Periodic acid – 0.5 gm
metabisulfite – 6 mL • Distilled water – 100 mL
• 1/N aqueous Solution Hydrochloric
acid – 5 mL
• Distilled water – 1000 mL
LUXOL FAST BLUE-PAS-HEMATOXYLIN
STAIN
FOR MYELIN

Results:
Myelin – blue green
Nuclei – dark blue
Capillaries - red
Cytoplasmic nucleoproteins – bluish
Notes and Special Considerations:
purple
FIXATION: 10% formalin
Fungi and PAS Positive elements –
SECTIONS:
rose to red
Celloidin sections
Paraffin
Frozen sections
WEIL’S METHOD
FOR MYELIN SHEATHS

• 4% Aqueous iron alum – 50 mL

Solution A

• 10% alcohol hematoxylin – 5 mL

• Distilled water – 45 mL
Solution B
(Alcohol
• Mix 50 mL solution A to solution B immediately before use
Hematoxylin)

• Weigert’s Borax Ferricyanide Solution

• Borax – 2 gm
• Potassium ferricyanide – 2.5 gm
Differentiator
• Distilled water – 200 mL
 Notes and Special Considerations:
WEIL’S METHOD The mordant and dye are mixed in the staining
FOR MYELIN SHEATHS solution. This is good method for FROZEN
SECTIONS.
As with hematoxylin stains, acid alcohol can be
used to differentiate the sections.

Notes and Special Considerations:

Fixation:
• Formol saline
• Formol calcium
Sections:

Result • Paraffin (10-15 um)

• Frozen (20-30 um)
• Myelin – black
Frozen Sections - are brought through
• Background – yellow
alcohols up to xylene and back again to
water.
STAINING OF ASTROCYTES
 Reactive astrocytosis or gliosis is the condition when astrocytes proliferate,
increase in size and develop more cytoplasm with prominent cytoplasmic
processes in response to local tissue injury.

 Reactive astrocytes stain well with PTAH; Normal astrocytes are poorly
stained with PTAH
 Cajal’s gold sublimate method - produce delicate staining of the
cytolasmic processes of normal and reactive astocytes

 Holzer technique/ Hortega lithium carbonate technique – demonstrate

gliosis
 Glial fibrillary acidic acid (GFAP)- immunohistochemical demonstration
of astrocytes
 CAJAL’S GOLD SUBLIMATE MET
FOR ASTROCYTES

Notes and Special

Staining Solutions: Results: Considerations:
1% Gold chloride – 5 mL Astrocytes – black Fixation:
5% Mercuric chloride – 5 Background – light brownish Cajal’s Formol Ammonium
mL Nerve cell – red Bromide for 2-25 days
Distilled water – 40 mL Nerve fibers - unstaineds (average 5 days)
Sections: Frozen Section
MODIFIED PTAH STAIN
FOR REACTIVE ASTROCYTES FOR NEUROGLIAL FIBERS
Reagents
• Acid Zenker’s Solution
• Phosphotungstic Acid Hematoxylin

Acid Zenker’s Solution

• Glacial Acetic Acid – 5 mL
• Zenker’s solution – 95 mL

Phosphotungstic Acid Hematoxylin

• Hematoxylin – 1 gm
• Phosphoptungstic acid – 20 gm
• Potassium permanganate – 0.2 gm
• Distilled water – 500 mL
• Adjust pH to 1.7 – 1.8 with 0.1 N hydrochloric acid or 0.1 N sodium
hydroxide
• Recheck pH immediately before each use and adjust accordingly
MODIFIED PTAH STAIN
FOR REACTIVE ASTROCYTES FOR NEUROGLIAL FIBERS

Notes and Special Considerations

Fixation:
10% buffered or unbuffered formalin
Sections:
Paraffin sections cut in 8 um

Potassium permanganate is use for ripening.

pH meter standardize with a known pH 2.0
bugger (HCl-KCl system)
MODIFIED HOLZER’S METHOD
FOR ASTROCYTIC PROCESSES (STEART 19880)

Mordant:
Chloroform – Alcohol mixture
1% phoshomolybdic acid – 10 mL
Chloroform – 160 mL
Absolute alcohol – 40 mL
Absolute Alcohol – 40 mL

Crystal Violet Stain:
Crystal violet – 2 gm
Absolute alcohol – 20 mL
Chloroform – 80 mL
Differentiating Solution
Aniline oil – 80 mL
Chloroform – 120 mL
Concentrated Ammonia – 10 drops
Filter before use.
MODIFIED HOLZER’S METHOD
FOR ASTROCYTIC PROCESSES (STEART 19880)

Notes and Special Considerations

Fixation:
Formalin
Helly’s fluid
Bouin’s fixative
Sections:
Paraffin sections (6-10 um)

Aniline oil is hazardous

STAINING OF OLIGODENDROCYTES
 Normal oligodendrocytes are
demonstrated by immuno-
histochemical methods using
antibodies to:
 Galactocerebroside

 Myelin basic protein

 Carbonic anhydrase-C
STAINING OF MICROGLIAL CELLS
 Derived from mononuclear
phagocytic system

 Appear as rod shape nuclei in H&E

 Resting or inactive Microglia

 Stain positively – CD68 antigens

 Stain faintly- leukocyte common

antigen (CD45)
THE END