HISTOLOGY

LECTURE # 29

INTRODUCTION TO SPECIAL STAINS TECHNIQUES:

NERVE TISSUE STAINING

Rationale: Special stain techniques are one of the major processes done in the histology laboratory.
These techniques are performed to be evaluated with the diagnostics slides from H&E’s. These stains are
used as an aid and a diagnostic tool for a final diagnosis.

Objective:
Once completed this lecture, the student should be able to:

a) Describe the different types of nerve tissue staining.

b) Learn the various methods of stain demonstration.

c) Learn the classification for Neurofibrils, axon, glial, astrocytes and myelin sheath staining.

d) Learn the procedures and diagnostic tools for each stain.

NERVE TISSUE

Introduction

• Nerve Tissue Nerve tissue can be divided into two anatomical parts:
– Central Nervous System (CNS)
• Brain & Spinal cord
– Peripheral Nervous System (PNS)
• All other nervous system

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 • In histology, nervous tissue consists of cells and cells processes and the various components of
nerve tissues. These normally fall in three groups:
– Neuronal cells and processes
– Glial cells and processes
– Myelin sheath

Neurons
1. Nissl Substances - basophilic material in the cytoplasm of the neuron
2. Nerve Cell Processes – Dendrites & Axons

Neuroglia
1. Oligodendroglia – small cells that function in the CNS to produce & probably maintain, the
myelin sheath surrounding many axons
2. Astrocytes – stellate cells of two types: Protoplasmic, which occurs in the gray matter, and
fibrous which occurs in the white matter
3. Microglia – fixed phagocytic cells found throughout the brain and spinal cord
4. Ependymal cells – true epithelial cells that line the ventricles and spinal canal, forms a barrier
between the cerebrospinal fluid and nervous tissue

Myelin

1. Is a complex, white, fatty, nonliving material containing protein, cholesterol, phospholipids, and
cerebrosides.

2. It is largely loss during routine paraffin processing with only neurokeratin, a resistant proteolipid, left
in the embedded block.

3. It is formed by oligodendroglia in the central nervous system and by Scwann cells in the peripheral
nervous system.

These are normally demonstrated by Luxol Fast Blue and Iron hematoxylin

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Nissl Substance: Cresyl Echt Violet Method (Luna, 1960)
I. Purpose of the stain: Identification of neurons in tissue sections, or demonstration of loss of
Nissl substances (chromatolysis).

II. Principle of the stain: They are very basophilic and will stain sharply with basic aniline dyes.
By varying the pH either Nissl substances can be stained by itself or nuclei and Nissl substances
can be stained.

III. Fixatives: 10% Neutral buffered formalin is preferred.

IV. Sectioning: Paraffin sections at 6-8 microns.

V. Controls: Section of spinal cord

VI. Reagents:
1. Cresyl Echt Violet Solution: Cresyl echt violet, distilled water. Need to be ripen
for 24 to 48 hors and filtered before use.
2. Balsam-Xylene Mixture: Canada Balsam & Xylene

VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Stain slides for 3-5 minutes in Cresyl Echt Violet solution. This is the step for the staining of
the nissl substances.
3. Rinse slides in two changes of distilled water.
4. Place sections in 95% alcohol for 30 seconds.
5. Place sections in absolute alcohol for 30 seconds.
6. Place section in xylene for 1 minute.
7. Place sections in balsam-xylene solution for 2 minutes.
8. Differentiate slides in two changes of absolute alcohol for 10 to 30 seconds each and check
microscopically.
9. Take through several changes of xylene. Steps 7 – 9 may have to be repeated constantly.
10. Coverslip with synthetic resin media.

VIII. Results:
Nissl Substances..………………………………………….. Blue to purple

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Nissl Substance: Cresyl Echt Violet Method (Vacca)

I. Purpose of the stain: Identification of neurons in tissue sections, or demonstration of loss of

Nissl substances (chromatolysis).

II. Principle of the stain: This procedure uses cresyl echt violet at an acid pH, restricting staining
to DNA- and RNA-containing structures.

III. Fixatives: 10% Neutral buffered formalin is preferred.

IV. Sectioning: Paraffin sections at 6-8 microns.

V. Controls: Section of spinal cord

VI. Reagents:
1. Cresyl Echt Violet Solution: Cresyl echt violet, distilled water. Need to be
ripen for 24 to 48 hors and filtered before use.
2. Balsam-Xylene Mixture: Canada Balsam & Xylene

VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Stain slides for 8 minutes in working Cresyl Echt Violet solution. This is the step for the
staining of the nissl substances.
3. Dehydrate sections in two changes of 95% alcohol & absolute alcohol.
4. Clear in two changes of xylene & coverslip.

Results: Nissl Substances & Nuclei ..………………………………………….. Blue purple

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 Nerve fibers, Nerve endings, and Neurofibrils
Bodian Method

I. Purpose of the stain: Staining of nerve fiber in tissue sections.

II. Principle of the stain: This procedure uses a silver proteinate known as Protargol™, which is
mixed in with several shots of copper creating a chemical reaction suitable to the staining of the
nerve fibers. Hydroquinone is the reducing reagent in this procedure.

III. Fixatives: 10% Neutral Buffered Formalin is preferred

IV. Sectioning: Cut Paraffin sections from 6 to 8 microns.

V. Controls: Best control is peripheral nerve or cerebral cortex. Spinal cord is not a good control,
since it will appear in a cross section.

VI. Reagents:
1. 1% Protargol™: Protargol™ & Distilled water.
2. Reducing solution: hydroquinone, Formaldehyde & distilled water.
3. 1% Gold chloride solution: gold chloride & distilled water.
4. 5% Sodium thiosulfate (Hypo): sodium hyposulfite & distilled water.
5. Aqua Regia: Hydrochloric acid concentrated, nitric acid concentrated. Prepare and use
under a hood.
6. Aniline blue solution: Aniline blue, oxalic acid, Phosphomolybdic acid & distilled water.

VII. Procedure:
1. Deparaffinize and hydrate sections to distilled water.
2. For every 100 mL of Protargol solution, add 4 to 6g of clean copper shot (cleaned with
aqua regia and rinsed well with distilled water). Place slides in this
solution and let stand at 37°C for 48 hours.
3. Rinse sections in three changes of distilled water.
4. Place slides in the reducing solution for 10 minutes.
5. Rinse in three changes of distilled water.
6. Tone sections in gold chloride solution for 10 minutes. This solution may be reused.
7. Rinse in three changes of distilled water.
8. Develop in oxalic acid solution, checking with the microscope, until the background is
gray and the nerve fibers appear clearly stained (approximately 3 to 5 minutes). Oxalic
acid treatment should not be prolonged since over treatment will ruin the silver proteinate
reaction.
9. Rinse in three changes of distilled water.
10. Treat sections with sodium thiosulfate for 5 minutes.
11. Rinse in distilled water.
12. Counterstain, if desired, with aniline blue solution (2 or 3 quick dips to give a light blue
background). See note 4.
13. Dehydrate in 95% alcohol and absolute alcohol, two changes each.
14. Clear in two changes of xylene.
15. Mount with synthetic resin.

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VIII. Results:
Nuclei……………………………..Black
Background.....................................Light Gray or Blue
Nerve Fibers................................... Black

Bodian Stain

Nerve Fibers

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Holmes Silver Nitrate Method

I. Purpose of the stain: The demonstration of nerve fibers and Neurofibrils in tissue section.

II. Principle of the stain: Holmes attributed inconsistent results obtained with the Bodian technique
to the fact that the Protargol solution never reaches the alkalinity necessary for optimal impreg-
nation, and he modified the technique by developing a buffered impregnating solution. The
pyridine in the solution is an alkali, and Holmes thought that this modified the electrostatic
condition of the tissue. This is an argyrophil silver method, requiring that chemical reduction be
used. The purposes of gold chloride, oxalic acid, and sodium thiosulfate are identified in the
description of the Bodian procedure.

III. Fixatives: 10% Neutral Buffered Formalin is preferred.

IV. Sectioning: Cut Paraffin sections from 10 to 15 microns.

V. Controls: Use a section of cerebral cortex. Spinal cord is not a good control for this stain as all
axon appear in cross section.

VI. Reagents:
a) Aqueous Silver Nitrate, 20%: Silver nitrate & Distilled water
b) Aqueous Silver Nitrate, 1 %: Silver nitrate: 20% Silver solution & Distilled water
c) Boric Acid Solution: Boric acid & Distilled water
d) Borax Solution: Sodium borate & distilled water
e) 10% Pyridine Solution: Pyridine & Distilled water
f) Impregnating Solution
1. Boric acid solution (fresh)
2. Borax solution (fresh)
3. Distilled water
4. Silver nitrate, 1 % aqueous solution
5. Pyridine, 10% aqueous solution
Mix boric acid solution and borax solution in a 500-mL flask. Add the water, then the aqueous
silver nitrate, and then the aqueous solution of pyridine. Mix thoroughly. Make enough solution
for 20 mL per slide.

g) Reducing Solution: Make fresh before use

1. Hydroquinone
2. Sodium sulfite (crystals)
3. Distilled water
h) Gold Chloride, 0.2%: Gold chloride, 1 % solution & Distilled water
i) Oxalic Acid Solution, 2%: Oxalic acid & Distilled water
j) Sodium Thiosulfate (Hypo): Sodium thiosulfate & Distilled water

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VII. Procedure:
1. Deparaffinize sections and hydrate to distilled water.
2. Place sections in 20% silver nitrate in the dark at room temperature for 1 hour.
3. Prepare the impregnating solution.
4. Take slides from 20% silver nitrate and wash for 10minutes in three changes of distilled water.
5. Place slides in impregnating solution, allowing at least 20 mL of solution per slide. Cover jar and
incubate overnight at 37°C.
6. Remove slides, shake off superfluous fluid, and place in the reducer for not less than 2 minutes.
7. Wash in running water for 3 minutes.
8. Rinse slides in distilled water.
9. Tone in 0.2% aqueous gold chloride for 3 minutes.
This solution may be reused until a brown precipitate forms or the solution becomes cloudy.
10. Rinse in distilled water.
11. Place slides in 2% aqueous oxalic acid for 3 to 10 minutes. When the axons are thoroughly
blue-black, stop the process.
12. Rinse in distilled water.
13. Place the slides in 5% aqueous sodium thiosulfate.
14. Wash in tap water for 10 minutes. As with Bodian stain, a counterstain may be applied at this
point.
15. Dehydrate in two changes each of 95% alcohol and absolute alcohol.
16. Clear in xylene and mount with synthetic resin.

VIII. Results:
Axon & Nerve Fibers……………………..Black
Neurofibrils.................................................Black

Holmes Silver Nitrate

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 Nerve Fibers, Neurofibrillary Tangles, and Senile Plaques:

Bielschowsky Method

I. Purpose of the stain: To demonstrate Nerve Fibers and the presence of neurofibrillary tangles
and senile plaques in Alzheimer’s disease.

II. Principle of the stain: The tissue is impregnated with the Ammoniacal silver solution. The silver
deposited on Neurofibrils and axons is then reduced to metallic silver by the formaldehyde in the
developer. Since the sections are not toned with gold chloride in this procedure, the yellow
background remains, Sodium thiosulfate removes and unreduced silver.

III. Fixatives: 10% Neutral Buffered Formalin.

IV. Sectioning: Cut Paraffin sections from 8 microns.

V. Controls: Tissue from the central nervous system must be used. If possible, the tissue should
contain senile plaques and neurofibrillary tangles.

VI. Reagents:
1. 1% Silver Nitrate: Silver nitrate & Distilled water: Prepare fresh.
2. 5% Silver Nitrate: Silver nitrate & Distilled water: Store in refrigerator.
3. 10%Nitric acid solution: Nitric acid & distilled water: Prepare fresh.
4. Developer:
a) Formaldehyde: 37 to 40%
b) Distilled water
c) Citric Acid
d) 10% Nitric acid
Prepare Fresh
5. 1% Ammonium hydroxide solution: Ammonium hydroxide, Conc. & Distilled water.
6. 2% Sodium thiosulfate (Hypo): sodium hyposulfite & distilled water.

VII. Procedure:
1. Deparaffinize the slides and hydrate to distilled water.
2. Place slides in 40 mL of 1.0% silver nitrate solution in a plastic Coplin jar and
microwave at power level 3 (180 W) for 1 minute. Dip the slides up and down several
times and allow them to remain in the warm solution (50°C) for 15 minutes.
3. Place the slides in distilled water.
4. Pour the warm 1 % silver nitrate used in step 2 into a 125-mL flask. Add 28% ammonium
hydroxide drop by drop with constant shaking, until the initial precipitate disappears and the
solution turns clear. Then add 5% silver nitrate drop by drop with constant shaking, until the
solution becomes slightly cloudy.
5. Pour the ammoniacal silver solution prepared in step 4 into a plastic Coplin jar. Place slides in
this solution and microwave at power level 3 (180 W) for 1 minute. Dip the slides up and down
several times and allow them to remain in the warm solution (60°C) for 15 minutes.
6. Place slides in 1 % ammonium hydroxide solution for not more than 20 seconds.
7. Add three drops of developer to the ammoniacal silver solution used in step 5.Quickly mix
with a glass rod and immediately place the slides in the solution for about 3 minutes or until the
tissue sections turn brown. The solution will turn a grayish color and a mirror of silver will
form on the sides of the Coplin jar and sometimes on the slides, but not on the tissue sections.
8. Place slides in 1 % ammonium hydroxide solution for not more than 15 seconds.
9. Rinse in three changes of distilled water.

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 10. Wipe off the mirror of silver from both sides of the slides, taking care not to damage
the tissue sections.
11. Place the slides in 2% sodium thiosulfate for 30 seconds.
12. Rinse slides in four changes of distilled water.
13. Dehydrate in 95% alcohol and absolute alcohol, two changes each.
14. Clear in two changes of xylene and mount with synthetic resin.

VIII. Results:
Axon…………………………………………...……………………..Brown to black
Cytoplasmic Neurofibrils…………………………………………… Brown to black
Neurofibrillary tangles and plaques of Alzheimer’s disease......…….Dark brown to black
Neuromelanin………………………………………………………. Black
Lipofuchsin…………………………………………………………. Brown to black

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Bielschowsky-PAS Method

I. Purpose of the stain: To demonstrate the presence of neurofibrillary tangles and senile plaques in
Alzheimer’s disease.

II. Principle of the stain: The tissue is impregnated with the Ammoniacal silver solution, and silver
is deposited on neurofibrils and axons. The silver is then reduced to metallic silver by the
formaldehyde in the developer. Gold chloride is used to tone the section, and this step eliminates
the yellow background. Sodium thiosulfate removes any unreduced silver. The Schiff’s reaction is
used to stain both basement membranes and Amyloid in the plaques.

III. Fixatives: 10% Neutral Buffered Formalin.

IV. Sectioning: Cut Paraffin sections from 8 to 10 microns.

V. Controls: Tissue from the central nervous system must be used. If possible, the tissue should
contain senile plaques and neurofibrillary tangles.

VI. Reagents:
1. 20% Silver Nitrate: Silver nitrate & Distilled water: Prepare fresh.
2. Ammoniacal Silver: 20% silver nitrate & Ammonium hydroxide drop by drop.
3. Developer:
a. Formaldehyde: 37 to 40%
b. Distilled water
c. Citric Acid
d. Nitric acid, Conc.
4. 0.5% Gold Chloride solution: Gold chloride & Distilled water.
5. 5% Sodium thiosulfate (Hypo): sodium hyposulfite & distilled water.
6. 1% Periodic Acid: Periodic acid & Distilled water.
7. Schiff’s Reagent

VII. Procedure:
1. Prepare the Ammoniacal silver solution before beginning the procedure.
2. Deparaffinize the slides and hydrate to distilled water.
3. Place slides in 20% silver nitrate in the dark at room temperature for 20 minutes.
4. Remove slides from the silver nitrate and wash once in distilled water.
5. Place slides in the Ammoniacal silver solution at room temperature for 20 minutes.
6. Wash slides in ammonia water (4 drops concentrated ammonium hydroxide to 100 ml of
distilled water).
7. While the slides are in the ammonia water, add 2 drops of developer to the Ammoniacal
silver solution used in step 5 and mix well.
8. Place slides in the mixed developer-ammoniacal silver solution. The tissue should turn
brown, average time is 3 minutes.
9. Wash well in ammonia water, then in distilled water.
10. Tone slides in gold chloride until the first gray appears, approximately 30 seconds.
11. Wash slides in ammonia water, then in distilled water for 1 minute.
12. Place slides in 5% sodium thiosulfate (hypo) for 30 seconds.
13. Wash slides in running tap water for 5 minutes.
14. Rinse sections well in distilled water.
15. Place slides in 1% periodic acid solution for 5 minutes.
16. Rinse slides well in two changes of distilled water.
17. Place sections in the Schiff’s reagent for 5 minutes.
18. Dehydrate slides in two changes of 95% alcohol and two changes absolute alcohol.

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 19. Clear in two changes of xylene and mount with synthetic resin.

VIII. Results:
Axon…………………………………………...……………………..Black
Amyloid(plaque cores and vascular)…………………………………Magenta
Neurofibrillary tangles and peripheral neurites or neuritic plaques.….Dark black
Lipofuchsin…………………………………………………….……. Magenta

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Sevier-Munger Method

I. Purpose of the stain: To demonstrate nerve fibers and the presence of neurofibrillary tangles and
senile plaques in Alzheimer’s disease.

II. Principle of the stain: The tissue is impregnated with the Ammoniacal silver solution. The silver
is deposited on neurofibrils and axons and then is reduced to metallic silver by formaldehyde.
Since the sections are not toned by gold chloride in this procedure, the yellow background
remains. Sodium thiosulfate removes any unreduced silver.

III. Fixatives: 10% Neutral Buffered Formalin.

IV. Sectioning: Cut Paraffin sections from 6 to 8 microns.

V. Controls: Tissue from the central nervous system must be used.

VI. Reagents:
1. 20% Silver Nitrate: Silver nitrate & Distilled water.
2. 10% Silver nitrate: Silver nitrate & Distilled water.
3. Formalin Solution: 37-40% Formaldehyde & Distilled water.
4. Sodium carbonate solution: sodium carbonate & Distilled water.
5. Ammoniacal Silver: 20% silver nitrate & Ammonium hydroxide drop by drop.
6. 5% Sodium thiosulfate (Hypo): sodium hyposulfite & distilled water.

VII. Reagents:
1. Deparaffinize the slides and hydrate to distilled water.
2. Preheat the 20% silver nitrate to 60oC for 15 minutes. Add the slides to the warm silver
solution and let them remain in the oven for 15 minutes.
3. Rinse one slide at a time in distilled water and place in a clean, dry staining jar.
4. While shaking gently, add 10 drops of the formalin solution to the working Ammoniacal
silver solution. Quickly pour this solution over the slides and let develop for 5 to 30 minutes
until golden brown. Check microscopically for completeness of the reaction. Do not wash.
Keep in motion during development to avoid precipitation.
5. Rinse slides well in three changes of fresh tap water.
6. Place in sodium thiosulfate solution for 2 minutes, and wash well in tap water.
7. Dehydrate, clear, and mount with synthetic resin.
VIII. Results:
Nerve endings…………………………………………...……………………..Black
Neurofibrillary tangles and peripheral neurites or neuritic plaques.………......Black

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 Glial Fibers

Mallory PTAH Stain

I. Purpose of the stain: The demonstration of glial fibers.

II. Principle of the stain: The amount of phosphotungstic acid is far greater than the amount of
hematein (20:1) in the staining solution, and it is believed that the tungsten binds all available
hematein to give a blue lake. This lake provides the blue color to selected tissue components (glial
fibers, nuclei, and to a certain extent, myelin). The red-brown- or salmon-colored components
(neurons) are believed to be stained by the phosphotungstic acid. Components will lose their red-
brown color after water or prolonged alcohol washing, so the dehydration steps following staining
should be rapid.

III. Fixatives: 10% Neutral Buffered Formalin.

IV. Sectioning: Cut Paraffin sections from 6 to 8 microns.

V. Controls: Use a section of cerebral cortex (not spinal cord) for the demonstration of glial fibers.

VI. Reagents:
1. PTAH Solution
a) Hematoxylin
b) Phosphotungstic acid
c) Distilled water
2. Lugol Iodine: Iodine & distilled water
Place the potassium iodide and about 150 mL of the water in a flask and stir until
dissolved. Dissolve the iodine in this concentrated solution of potassium iodide. When the
iodine is dissolved, add the remaining water and mix well.
3. 5% Sodium thiosulfate (Hypo): sodium hyposulfite & distilled water.
4. 1% Potassium Permanganate: Potassium permanganate & Distilled water.
5. 5% Oxalic Acid Solution: Oxalic acid & Distilled water.

VII. Reagents:
1. Deparaffinize the sections and hydrate to distilled water.
2. Mordant the sections overnight at room temperature in Zenker solution containing acetic acid.
3. Wash the sections in running water for 15 minutes.
4. Place in Lugol iodine for 15 minutes. Do not take the slides through sodium thiosulfate (hypo)
as this may impair the subsequent staining reaction.
5. Decolorize the sections in 95% alcohol for at least 1 hour.
6. Rinse rapidly in three changes of distilled water.
7. Place sections in 1 % potassium permanganate for 5 minutes.
8. Wash in running tap water for 10 minutes.
9. Decolorize the sections in 5% oxalic acid for 5 minutes.
10. Wash in running tap water for 10 minutes.
11. Stain in PTAH solution overnight at room temperature.
12. Dehydrate rapidly through two changes each of 95% and absolute alcohols, clear in xylene,
and mount with synthetic resin.

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VIII. Results:
Glial fibers….…………………………………………...……………………..Blue
Nuclei………………………………………………………………………….Blue
Neurons………………………………………………………………………..Salmon
Myelin…………………………………………………………………………Blue

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Holzer Method

I. Purpose of the stain: The demonstration of glial fibers and areas of gliosis.

II. Principle of the stain: Glial fibers are stained with crystal violet and are resistant to
decolorization with the alkaline aniline-chloroform mixture.

III. Fixatives: 10% Neutral Buffered Formalin.

IV. Sectioning: Cut Paraffin sections from 6 to 8 microns.

V. Controls: Use a section of cerebral cortex (not spinal cord) for the demonstration of glial fibers.

VI. Reagents:
1. Aqueous Phosphomolybdic Acid Solution: Phosphomolybdic acid & Distilled water. Prepare
fresh.
2. Phosphomolybdic alcohol solution: .5% Phosphomolybdic acid & 95% alcohol.
3. Absolute alcohol-Chloroform Mixture: Absolute alcohol & Chloroform. Prepare fresh.
a. Crystal Violet Stain, Prepare fresh:
b. Crystal violet
c. Absolute alcohol
4. Chloroform
5. Potassium bromide solution: potassium bromide & Distilled water.
6. Differentiating solution, Prepare fresh:
a. Aniline oil
b. Chloroform
c. Ammonium hydroxide, concentrated

VII. Procedure:
1. Deparaffinize the slides and hydrate to distilled water.
2. Place the sections in fresh phosphomolybdic acid-alcohol for 3 minutes.
3. Drain off the excess fluid, place slides on a staining rack, and cover the sections with absolute
alcohol-chloroform mixture. The tissue should become translucent.
4. While the sections are still wet, cover them with crystal violet stain and allow to remain for 30
seconds.
5. Replace the stain with 10% Potassium bromide, washing for 1 minute with this solution.
6. Blot the section dry and then allow them to air-dry thoroughly.
7. Differentiate slides individually for 30 seconds in the differentiating solution.
8. Wash in several changes of xylene. Steps 7 & 8 may have to repeated several times until the
background is very pale blue or colorless.
9. Mount with synthetic resin.
VIII. Results:
Glial fibers….……...…..Blue
Background……………Very pale blue to colorless

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Astrocytes: Cajal Stain

I. Purpose of the stain: The demonstration of astrocytes. This method has been replaced to a great
extent by immunohistochemical procedures.

II. Principle of the stain: Astrocytes are selectively stained with the Cajal gold sublimate method on
frozen sections.

III. Fixatives: Formalin ammonium bromide for no less than 2 days and no more than 25 days. If the
tissue has been fixed originally in 10% neutral buffered formalin, wash and place in formalin
ammonium bromide for 48 hours before proceeding with the technique.

IV. Sectioning: Cut frozen sections at 20-30 microns. Do not pick up on slides; the section should be
free-floating for this technique. Tissue will section better if washed in tap water for 30 minutes
before freezing.

V. Controls: Use a section of cerebral cortex (not spinal cord) for the demonstration of astrocytes.

VI. Reagents:
1. Formalin Ammonium Bromide: Ammonium bromide, Formaldehyde 37-40% & Distilled
water.
2. Gold Sublimate: 1% Gold Chloride, 1% Mercuric chloride & Distilled water.
3. 5% Sodium Thiosulfate (Hypo): Sodium thiosulfate & Distilled water.

VII. Procedure:
1. Wash the free-floating frozen section in several changes of distilled water.
2. Transfer the sections to gold sublimate solution, and leave in the dark for 4 hours. The
sections should be purple.
3. Wash well in distilled water.
4. Treat with 5% Sodium thiosulfate for 2 minutes.
5. Wash section well in several changes of distilled water.
6. Carefully mount the sections on slides, blot with bibulous paper, and dehydrate in 95% &
100% alcohols.
7. Clear in xylene and mount in synthetic resin.
VIII. Results:
Astrocytes with perivascular feet….…………………………………………………..Black

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Myelin Sheath: Weil Method

I. Purpose of the stain: The demonstration of myelin in tissue. When an axon degenerates, the
myelin sheath breaks down into simpler lipids; these simple lipids will be removed eventually.

II. Principle of the stain: The mordant-hematoxylin solution attaches to the phospholipid component
of the myelin sheath, which has an affinity for the cationic dye lake. This is a regressive staining
technique with differentiation usually accomplished in two steps. The first differentiation is
accomplished macroscopically with ferric ammonium sulfate (excess mordant), which removes
most of the excess dye. The second differentiation is done microscopically with borax ferricyanide
(oxidizer), which removes any remaining nonspecifically bound hematoxylin lake and forms a col-
orless oxidation product. Only the myelin sheath and red blood cells are left stained.

III. Fixatives: 10% Neutral buffered formalin.

IV. Sectioning: Cut paraffin section between 15 to 30 microns.

V. Controls: Use a section of spinal cord or medulla.

VI. Reagents:
1. Ferric Ammonium Sulfate Solution, 4%: Ferric ammonium sulfate & Distilled water
2. Alcoholic Hematoxylin, 10%: Hematoxylin powder & Absolute alcohol.
3. This solution should be allowed to stand for 2 to 3 days, but prolonged ripening is
unnecessary, because the iron used in the staining solution is a strong oxidizer.
4. Staining Solution: Hematoxylin, 10% alcohol solution, distilled water.
5. Mix in an Erlenmeyer flask and add: Ferric ammonium sulfate, 4% solution. Prepare fresh.
6. Differentiating Solution: Sodium borate, Potassium ferricyanide & Distilled water.

VII. Procedure:
1. Deparaffinize sections and hydrate to distilled water.
2. Transfer sections to the staining solution and stain for 30 minutes at 54°C to 56°C.
3. Wash in two changes of tap water.
4. Differentiate in 4% ferric ammonium sulfate until the gray matter can just be distinguished
from the white matter and the stain is removed from the slides.
5. Wash in three changes of tap water.
6. Complete differentiation of the sections in sodium borate-potassium ferricyanide solution. This
differentiation should be controlled microscopically until the gray and white matters are sharply
defined.
7. Wash sections in two changes of tap water.
8. Treat sections with diluted ammonia water (about 6 drops to 100 mL of water).
9. Wash in distilled water.
10. Dehydrate in two changes each of 95% and absolute alcohols.
11. Clear in xylene and mount with synthetic resin.

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VIII. Results:
Myelin sheath. . . . . . . . . . . . . . . . . Blue to blue-black
Background. . . . . . . . . . . . . . . . . . Light tan

Myelin Sheath: Luxol Fast Blue

1. Purpose of the stain: The demonstration of myelin in tissue. When an axon degenerates, the
myelin sheath breaks down into simpler lipids; these simple lipids will be removed eventually.

2. Principle of the stain: Luxol fast blue, like alcian blue, is of the sulfonated copper
phthalocyanine type, but is alcohol soluble, whereas alcian blue is water-soluble. Staining is due
to lipoproteins, and the mechanism is one of an acid-base reaction with salt formation; the base
of the lipoprotein replaces the base of the dye.

3. Fixatives: 10% Neutral buffered formalin.

4. Sectioning: Cut paraffin section between 10 to 15 microns.

5. Controls: A section of spinal cord or medulla provides a good control.

6. Reagents:
a) 0.1% Luxol fast blue: Luxol fast blue MBSN, 95% alcohol: Dissolve the dye in alcohol,
then add: 10% Acetic acid.
b) 0.05% Lithium carbonate: Lithium carbonate& Distilled water.
c) 70% Alcohol solution: Absolute alcohol & Distilled water.

7. Procedure:
1. Deparaffinize sections and hydrate to distilled water.
2. Place slides in Luxol fast blue solution and leave overnight at 56°C to 58°C. The container
should be tightly capped as this alcoholic solution will evaporate readily.
3. Rinse sections in 95% alcohol to remove excess stain.
4. Rinse in distilled water.
5. Begin the differentiation by immersing the slides in lithium carbonate solution from 10 to 20
seconds.
6. Continue the differentiation in 70% alcohol solution until the greenish blue of the white matter
19
 contrast sharply with the colorless gray matter.
7. Wash sections in distilled water.
8. Dehydrate in two changes each of 95% and absolute alcohols.
9. Clear in xylene and mount with synthetic resin.
8. Results:
Myelin sheath. . . . . . . . . . . . ……………………………………………………….. . . . .Blue
Background. . . . . . . ……………………………………………………….. . . . . . . . . . . Colorless

20
Myelin Sheath and Nissl Substances Combined Stain:
Luxol Fast Blue-Cresyl Echt Violet

I. Purpose of the stain: The demonstration of both myelin and Nissl substances in tissue sections.
Nissl substances are lost after cell injury, and if the axon degenerates, the myelin covering also
breaks down.

II. Principle of the stain: Luxol fast blue, like alcian blue, is of the sulfonated copper phthalocyanine
type, but is alcohol soluble, whereas alcian blue is water-soluble. Staining is due to lipoproteins,
and the mechanism is one of an acid-base reaction with salt formation; the base of the lipoprotein
replaces the base of the dye. They are very basophilic and will stain sharply with basic aniline
dyes. By varying the pH either Nissl substances can be stained by itself or nuclei and Nissl
substances can be stained.

III. Fixatives: 10% Neutral buffered formalin.

IV. Sectioning: Cut paraffin section between 10 to 15 microns.

V. Controls: A section of spinal cord or medulla provides a good control.

VI. Reagents:
a) 0.1% Luxol fast blue: Luxol fast blue MBSN, 95% alcohol: Dissolve the dye in alcohol,
then add: 10% Acetic acid.
b) 0.05% Lithium carbonate: Lithium carbonate& Distilled water.
c) 70% Alcohol solution: Absolute alcohol & Distilled water.
d) 10% Acetic acid: Acetic acid & Distilled water.

e) Cresyl Echt Violet: Cresyl echt violet & Distilled water. Just before use add 15 drops
of 10% Acetic acid, filter and pre-heat. This solution is not very stable.

VII. Procedure:
1. Deparaffinize sections and hydrate to distilled water.
2. Place slides in Luxol fast blue solution and leave overnight at 56°C to 58°C. The container
should be tightly capped as this alcoholic solution will evaporate readily.
3. Rinse sections in 95% alcohol to remove excess stain.
4. Rinse in distilled water.
5. Begin the differentiation by immersing the slides in lithium carbonate solution from 10 to 20
seconds.
6. Continue the differentiation in 70% alcohol solution until gray matter and white matter can be
distinguished. Do not overdifferentiate.
7. Wash sections in distilled water.
8. Finish differentiating the sections by rinsing them briefly in lithium carbonate solution, then
putting them in 70% alcohol. The greenish blue of the white matter contrast sharply with the
colorless gray matter.
9. Rinse slides thoroughly in distilled water.
10. Place slides in Cresyl echt violet solution for 6 minutes. Filter and preheat cresyl echt violet
solution to 57°C just before use. Keep hot during staining.
11. Differentiate in several changes of 95% alcohol.
12. Dehydrate in two changes each of 95% and absolute alcohols. Clear in xylene and mount with
synthetic resin.
VIII. Results:
Myelin sheath. . . . . . . . . . . . ……………………………………………………….. . . . .Blue
Nissl Substances…………………………………………………………………………..Violet
Nuclei……………………………………………………………………………………...Violet
21
22
Myelin Sheath and Nerve Fibers Combined Stain:
Luxol Fast Blue-Holmes Silver Nitrate Method

I. Purpose of the stain: The demonstration of both myelin and nerve fibers in the same tissue
section. If the axon degenerates, the myelin covering also breaks down into simpler lipids that are
eventually removed.

II. Principle of the stain: Luxol fast blue, like alcian blue, is of the sulfonated copper phthalocyanine
type, but is alcohol soluble, whereas alcian blue is water-soluble. Staining is due to lipoproteins,
and the mechanism is one of an acid-base reaction with salt formation; the base of the lipoprotein
replaces the base of the dye.

Holmes attributed inconsistent results obtained with the Bodian technique to the fact that the
Protargol solution never reaches the alkalinity necessary for optimal impregnation, and he
modified the technique by developing a buffered impregnating solution. The pyridine in the solu-
tion is an alkali, and Holmes thought that this modified the electrostatic condition of the tissue.
This is an argyrophil silver method, requiring that chemical reduction be used. The purposes of
gold chloride, oxalic acid, and sodium thiosulfate are identified in the description of the Bodian
procedure.

III. Fixatives: 10% Neutral buffered formalin.

IV. Sectioning: Cut paraffin section between 10 to 15 microns.

V. Controls: A section of cerebral cortex. A spinal cord is NOT good for this procedure due to the
cross section of the axons.

VI. Reagents:
1. 0.1% Luxol fast blue: Luxol fast blue MBSN, 95% alcohol: Dissolve the dye in alcohol, then
add: 10% Acetic acid.
2. 0.05% Lithium carbonate: Lithium carbonate& Distilled water.
3. 70% Alcohol solution: Absolute alcohol & Distilled water.
4. 10% Acetic acid: Acetic acid & Distilled water.
5. Aqueous Silver Nitrate, 20%: Silver nitrate & Distilled water
6. Aqueous Silver Nitrate, 1 %: Silver nitrate: 20% Silver solution & Distilled water
7. Boric Acid Solution: Boric acid & Distilled water
8. Borax Solution: Sodium borate & distilled water
9. 10% Pyridine Solution: Pyridine & Distilled water
10. Impregnating Solution
a. Boric acid solution (fresh)
b. Borax solution (fresh)
c. Distilled water
d. Silver nitrate, 1 % aqueous solution
e. Pyridine, 10% aqueous solution
Mix boric acid solution and borax solution in a 500-mL flask. Add the water, then the aqueous
silver nitrate, and then the aqueous solution of pyridine. Mix thoroughly. Make enough solution
for 20 mL per slide.

23
 11. Reducing Solution: Make fresh before use
a. Hydroquinone
b. Sodium sulfite (crystals)
c. Distilled water
12. Gold Chloride, 0.2%: Gold chloride, 1 % solution & Distilled water
13. Oxalic Acid Solution, 2%: Oxalic acid & Distilled water
14. Sodium Thiosulfate (Hypo): Sodium thiosulfate & Distilled water.

VII. Procedure:
1. Deparaffinize sections and hydrate to distilled water.
2. Place sections in 20% silver nitrate in the dark at room temperature for 1 hour.
3. Prepare the impregnating solution.
4. Take slides from 20% silver nitrate and wash for 10minutes in three changes of distilled water.
5. Place slides in impregnating solution, allowing at least 20 mL of solution per slide. Cover jar and
incubate overnight at 37°C.
6. Remove slides, shake off superfluous fluid, and place in the reducer for not less than 2 minutes.
7. Wash in running water for 3 minutes, the rinse slides in distilled water.
8. Tone in 0.2% aqueous gold chloride for 3 minutes.
This solution may be reused until a brown precipitate forms or the solution becomes cloudy.
9. Rinse in distilled water.
10. Place slides in 2% aqueous oxalic acid for 3 to 10 minutes. When the axons are thoroughly
blue-black, stop the process.
11. Rinse in distilled water.
12. Place the slides in 5% aqueous sodium thiosulfate.
13. Wash in tap water for 10 minutes.
14. Place the slides briefly in 95% alcohol briefly.
15. Place slides in Luxol fast blue solution and leave overnight at 56°C to 58°C. The container
should be tightly capped as this alcoholic solution will evaporate readily.
16. Rinse sections in 95% alcohol to remove excess stain.
17. Place slides in distilled water.
18. Place the slides in 0.05% lithium carbonate for 15 seconds.
19. Differentiate slides in 70% alcohol for 20 to 30 seconds.
20. Rinse in distilled water. Repeat steps 18 to 20 if Luxol fast blue needs more differentiation.
21. Dehydrate in two changes each of 95% and absolute alcohols.
22. Clear in xylene and mount with synthetic resin.
VIII. Results:
Myelin sheath. . . . . . . . . . . . ………………………………………………………..Blue to green
Axon and nerve fibers……………………………………………………..………...Black

24
Stain Bodian Method Holmes Silver Nitrate PTAH Holzer’s Method
Method
Fixative 10% Neutral 10% Neutral Buffered 10% Neutral Buffered 10% Neutral Buffered
Buffered Formalin Formalin Formalin
Formalin
Section 6-8µ 10-15µ 6-8µ 6-8µ

Control Peripheral nerve or Cerebral cortex Cerebral cortex Cerebral cortex

cerebral cortex
Reagents Protargol™ Silver Nitrate Hematoxylin Phosphomolybdic acid
Hydroquinone Boric acid Phosphotungstic acid 95% Alcohol
Formaldehyde Sodium borate Lugol’s Iodine Chloroform
Gold chloride Pyridine Oxalic acid Crystal violet
Hypo Hydroquinone Sodium hyposulfite Potassium bromide
Hydrochloric acid Sodium sulfite Potassium permanganate Aniline oil
Nitric acid Gold chloride Ammonium hydroxide
Aniline blue Oxalic acid
Oxalic acid Sodium hyposulfite
Phosphomolybdic acid

Results Nerve Fibers – Black Axons – Black Glial fibers – Blue Glial fibers – Blue
Background – Blue Nerve Fibers – Black Nuclei-Blue
Nuclei - Black Neurofibrils - Black Neuron-salmon
Myelin-Blue

25
Stain Cajal’s Stain Weil’s Method Luxol Fast Blue Luxol Fast Blue/Cresyl
Echt Violet

Fixative Formalin Ammonium 10% Neutral Buffered 10% Neutral 10% Neutral Buffered
Bromide Formalin Buffered Formalin
Formalin
Section 10-30µ frozen sections 10-15µ 10-15µ 10-15µ

Control Cerebral cortex Spinal cord or Medulla Spinal cord or Spinal cord or Medulla
Medulla
Reagents Ammonium bromide Ferric ammonium sulfate Luxol fast blue Luxol fast blue
Formaldehyde Hematoxylin Acetic acid Acetic acid
Gold chloride Absolute alcohol Lithium carbonate Lithium carbonate
Mercuric chloride Sodium borate Absolute alcohol Absolute alcohol
Hypo Potassium ferrocyanide Cresyl echt violet

Results Astrocytes- Black Myelin Sheaths-blue to Myelin-Blue Myelin – Blue

blue-black Nissl Substances-violet
Nuclei-violet

26