Perls' Prussian Blue

for Hemosiderin

steps

materials

print

perls-prussian-blue-staining-lung-tissue-pulmonary-veno-occlusive-disease-600x400

Figure 1. Perls' Prussian Blue Staining of Lung Tissue with Pulmonary Veno-occlusive Disease

Perls' Prussian blue staining reveals extensive intra-alveolar hemosiderin deposition following pulmonary hemorrhage in pulmonary veno-occlusive disease. Intra-alveolar hemosiderin deposition Case 101 by Yale Rosen is licensed under CC BY-SA 2.0.

Materials

Neutral red
Stock solution A
Material Amount
Potassium ferrocyanide 2 g
Distilled water 100 mL
Stock solution B
Material Amount
Hydrochloric acid, conc. 2 mL
Distilled water 100 mL
Working solution
Material Amount
Stock solution A 1 volume
Stock solution B 1 volume

Material	Amount
Potassium ferrocyanide	2	g
Distilled water	100	mL

Material	Amount
Hydrochloric acid, conc.	2	mL
Distilled water	100	mL

Material	Amount
Stock solution A	1	volume
Stock solution B	1	volume

Make the working solution immediately before use. Do not use if more than 30 minutes old.

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Avoid iron containing materials and jars while fixing as these may contaminate the tissue. Acid containing fixatives may remove some of the iron deposits, but apart from that most are satisfactory.

Protocol

Bring sections to distilled water with xylene and ethanol.
Place into the working solution for 15 minutes.
Rinse with distilled water, then tap water.
Stain with neutral red for one minute.
Rinse well with tap water.
Dehydrate with ethanol.
Clear with xylene.

Expected Results

Ferric iron – blue
Nuclei – red

Notes

Avoid washing with tap water before placing into the working solution, as rust in the water or tap fixtures could cause false positive staining.
Wash well at step 3, as traces of iron will form a granular red deposit with neutral red.
Iron ores can be demonstrated, but the acid concentration in solution A may need to be increased to 10% or more.
Hemosiderin forms the body’s iron stores. The iron is in the ferric state, and may be demonstrated by releasing it from hemosiderin with hydrochloric acid, forming ferric chloride. The iron reacts with potassium ferrocyanide
to form ferric ferrocyanide. This is an insoluble, blue compound known as Prussian blue or Berlin blue. The intensity of the colour gives some indication as to amount, but it is qualitative only. Other sources of ferric iron will also be demonstrated.
The reaction is: 4FeCl₃+ 3K₄Fe(CN)₆ = Fe₄[Fe(CN)₆]₃ + 12KCl

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Culling, C.F.A., Allison, R.T. and Barr, W.T.
Cellular Pathology Technique, Ed.4.
Butterworth, London, UK.
Susan Budavari, Editor, (1996)
The Merck Index, Ed. 12
Merck & Co., Inc., Whitehouse Station, NJ, USA

March 30, 2023

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Lendrum, Slidders & Fraser’s Trichrome for Collagen and Other Tissues

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Lendrum, Slidders & Fraser's Trichrome

for Collagen and Other Tissues

steps

materials

print

Materials

Small Molecular Weight (MW) Solution

Material	Amount
Dye	0.5	g
Phosphotungstic acid	2	g
Ethanol, 95%	100	mL

Use one of the dyes from the appropriate list below:

Small MW Dyes

Dye	CI Name	Author’s Code
Martius yellow	Acid yellow 24	AY24
Orange G	Acid orange 10	A010
Azo-eosin	Acid red 4	AR4
Propalan red 3GX	Acid red 57	AR57
Acid fuchsin	Acid violet 19	AV19

Large MW solution

Material	Amount
Dye	0.5	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Use one of the dyes from the appropriate list below:

Large MW Dyes

Dye	CI Name	Author’s Code
Sun yellow	Direct yellow 11	DY11
Sirius red F3B	Direct red 80	DR80
Benzo new blue GS	Direct blue 10	DR10
Durazol brilliant blue B	Direct blue 109	DB109

Tissue Sample

3-5µ paraffin sections of formal sublimate fixed tissue is preferred. If formalin fixation is used, the sections may require secondary fixation with picro-mercuric-alcohol.

Protocol

Bring sections to water via xylene and ethanol.
Stain nuclei with an acid resistant nuclear stain.
Rinse with 95% ethanol.
Place into small MW solution for 3 minutes.
Rinse with distilled water.
Place in the large MW solution for 30 minutes to 2 hours.
Rinse with water.
Dehydrate with absolute ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

The results vary depending on the particular combination of small and large molecular weight dyes chosen. As a general guide, the small molecular weight dye can be expected to stain erythrocytes, cytoplasm and muscle. The large molecular weight dye can be expected to stain collagen. The authors particularly recommended the following combinations:

General use – AO10, DR80
After PAS – AY24, DY11
After silver – AR4, DY11

Notes

This technique has not gained favour as it uses some difficult to obtain dyes.
Picro-mercuric-alcohol is a saturated solution of both picric acid and mercuric chloride in absolute ethanol. It is usually applied to sections overnight after dewaxing and treating with ethanol, but before washing with water. If used, the iodine-thiosulphate sequence should be used to remove mercury pigment before staining.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Lendrum, A.C., Slidders, W. and Fraser, S., (1972),
Renal hyalin: A study of amyloidosis and diabetic fibrinous vasculosis with new staining methods.,
Journal of Clinical Pathology, v 25, page 373

March 30, 2023

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Shoobridge’s Polychrome for Collagen and Other Structures

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Shoobridge's Polychrome

for Collagen and Other Structures

steps

materials

print

Materials

Lillie’s alum hematoxylin
Iron alum
Material Amount
Ferric ammonium sulphate 25 g
Distilled water 500 mL
Glycerol 70 mL
This should be pH 2.0.
Acid ethanol
Material Amount
Ethanol, absolute 100 mL
Distilled water 100 mL
Hydrochloric acid, concentrated 1 mL
Formal primer
Material Amount
Picric acid, saturated aqueous 400 mL
Formalin, concentrated 100 mL
This should be pH 2.0.
Naphthol yellow primer
Material Amount
Naphthol yellow S 1 g
Distilled water 400 mL
Adjust to pH 2.0 with HCl or NAOH.
Tungsto-orange
Material Amount
Phosphotungstic acid 0.5 g
Distilled water 200 mL
Orange G 1 g
Preparation
1. Dissolve the phosphotungstic acid.
2. Then add the orange G.
3. Adjust to pH 2.5 with HCl or NaOH.
Tungsto-acid fuchsin
Material Amount
Phosphotungstic acid 0.5 g
Distilled water 200 mL
Acid fuchsin 1 g
Preparation
1. Dissolve the phosphotungstic acid.
2. Then add the acid fuchsin.
3. Adjust to pH 2.5 with HCl or NaOH
Tungsto-methyl blue
Material Amount
Phosphotungstic acid 0.5 g
Distilled water 200 mL
Methyl blue 1 g
Preparation
1. Dissolve the phosphotungstic acid.
2. Then add the acid fuchsin.
3. Adjust to pH 2.5 with HCl or NaOH

Material	Amount
Ferric ammonium sulphate	25	g
Distilled water	500	mL
Glycerol	70	mL

Material	Amount
Ethanol, absolute	100	mL
Distilled water	100	mL
Hydrochloric acid, concentrated	1	mL

Material	Amount
Picric acid, saturated aqueous	400	mL
Formalin, concentrated	100	mL

Material	Amount
Naphthol yellow S	1	g
Distilled water	400	mL

Material	Amount
Phosphotungstic acid	0.5	g
Distilled water	200	mL
Orange G	1	g

Material	Amount
Phosphotungstic acid	0.5	g
Distilled water	200	mL
Acid fuchsin	1	g

Material	Amount
Phosphotungstic acid	0.5	g
Distilled water	200	mL
Methyl blue	1	g

Tissue Sample

5µ paraffin sections of tissues fixed in neutral buffered formalin for 6-24 hours as the primary fixative, followed by secondary fixation in 0.5% aqueous mercuric chloride for 30 minutes at 37°C, or for several hours at room temperature. Treatment with mercuric chloride may be omitted but the staining results will be inferior.

Protocol

Bring sections to water via xylene and ethanol.
Remove mercury pigment and wash well with tap water.
Place into iron alum for 10 minutes.
Rinse well with tap water.
Rinse with distilled water.
Place into Lillie’s hemalum for 10 minutes.
Rinse well with tap water.
Differentiate with acid ethanol for 15-30 seconds.
Blue with running tap water (50°C). Avoid alkaline blueing solutions.
Rinse with distilled water (50°C).
Stain with naphthol yellow primer or picro-formal primer for 30 minutes at 50°C.
Prewarm the solution.
Wash in running tap water until differentiated (about 5 minutes at pH 6.5-7.0). Only erythrocytes, muscle and fibrin should be yellow.
Stain in tungsto-orange solution for 5 minutes at room temperature.
Rinse with tap water (pH 7.0 or less) for 15-30 seconds.
Stain with tungsto-acid fuchsin for 5 minutes at room temperature.
Rinse with tap water (pH 7.0 or less) for 15-30 seconds.
Stain with tungsto-methyl blue for 5 minutes at room temperature.
Rinse with tap water (pH 7.0 or less) for 15-30 seconds.
Drain excess water.
Dehydrate rapidly with 3 changes of absolute ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Erythrocytes – Yellow
Striated muscle – Yellow, or bright red if the primer is omitted
Fibrin – Varies: unstained, yellow-red, scarlet, magenta, blueish-red as it ages.
Fibrinoid – Varies: yellow-red, scarlet, crimson.
Albumin – Bright orange-red
Elastin – Clear pink-red
Nucleoli – Bright red
Platelets: Fresh to medium – Unstained to brick red
Platelets: Old – Purplish, grey or pale blue
Secretory granules (e.g. pituitary) – Yellow, red or blue
Thyroid colloid – Red or blue (inactive tends to be blue)
Bile – Emerald to olive green
Primary amyloid – Smoky blue
Secondary amyloid – Pale blue
Most mucins – Pale blue
Reticulin – Blue
Collagen – Deep Blue
Basement membrane – Intense deep Blue

Notes

It is strongly advised that the original paper be consulted as it contains a great deal of precise information.
The naphthol yellow primer was recommended for routine use, as the picro-formal primer gave redder results.
The original paper includes a one-step method using the same dyes. Three stock solutions are prepared. Each solution contains 2 g of dye dissolved into 100 mL of 1% phosphotungstic acid. The staining solution is made by combining equal parts of these three solutions. Staining times are not given and should be established by trial and error.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Shoobridge, Michael P. K., (1983),
A new principle in polychrome staining: A system of automated staining, complementary to hematoxylin and eosin, and usable as a research tool.,
Stain Technology, v 58, page 245

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Cook’s Copper Hematoxylin

By Carmen WongDye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type

Protocol

Cook’s Copper Hematoxylin

steps

materials

print

Materials

Material	Amount	Function
Hematoxylin	10 g	Dye
Cupric sulfate	2.5 g	Mordant
Potassium aluminum sulfate	15 g	Mordant
Distilled water	100 mL	Solvent

Compounding procedure

Place the dry powders in a mortar and grind together.
Add sufficient water to make a paste.
Leave for 48 hours, then add the remaining water.
Leave for a further 12 hours, and filter.

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for an appropriate time.
Rinse with water.
Differentiate if necessary
Wash well with water.
Counterstain if wished.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue-black
Background – as counterstain or unstained

Notes

The staining time should be determined by trial.
The original method called for 15 grams of “extract of logwood”. Gray comments that this has about 70% dye content, so it has been specified to use 10 grams of dye.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Hogg, J. (1883)
The microscope
Routledge, London, England

March 30, 2023

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Howell’s Rubeanic Acid for Copper Deposits

By Carmen WongMetal Impregnation, Metal Impregnation, Non-Silver, Protocols, Stain Type

Protocol

Howell's Rubeanic Acid

for Copper Deposits

steps

materials

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Materials

Eosin Y, 0.1% in absolute ethanol.
Sodium acetate, 10% aqueous.
Stock rubeanic acid
Material Amount
Rubeanic acid 50 mg
Ethanol, absolute 50 mL
Working rubeanic acid
Material Amount
Stock rubeanic acid 2.5 mL
Sodium acetate, 10% aqueous. 50 mL

Material	Amount
Rubeanic acid	50	mg
Ethanol, absolute	50	mL

Material	Amount
Stock rubeanic acid	2.5	mL
Sodium acetate, 10% aqueous.	50	mL

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are satisfactory.

Protocol

Bring sections to water via xylene and ethanol.
Place in the working rubeanic acid solution overnight at 37°C.
Place into 70% ethanol for 15 minutes.
Place into absolute ethanol for 6 hours.
Lightly counterstain with alcoholic eosin.
Rinse well with absolute ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Copper – green-black granules
Cytoplasm – pink

Notes

Use both negative and positive controls, as occasional failures in staining occur. A suitable positive control is a liver section from a known case of Wilson’s disease. Failing that, copper may be found in liver from chronic active hepatitis.
Copper forms a chelate with rubeanic acid, as do some other metals. Of these, cobalt and nickel may be confused with copper, so sodium acetate is added to the solution to inhibit them.
Rubeanic acid is also known as ethanedithioamide, dithiooxamide or dithiooxalic diamide, with the formulae below. It would appear that the copper is chelated between the sulphur and nitrogen (see Kiernan).

Rubeanic chemical structure

H₂NCSCSNH₂

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Bancroft, J.D. and Stevens A. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.
Susan Budavari, Editor, (1996)
The Merck Index, Ed. 12
Merck & Co., Inc., Whitehouse Station, NJ, USA
Kiernan. J.A., (1999)
Histological and histochemical methods: Theory and practice, Ed. 3
Butterworth Heinemann, Oxford, UK.

March 30, 2023

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Faure’s Iron Hematoxylin

By Carmen WongDye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type

Protocol

Faure's Iron Hematoxylin

steps

materials

print

Materials

Solution A

Material	Amount	Function
Hematoxylin	3.2 g	Dye
90% ethanol	100 mL	Solvent

Solution B

Material	Amount	Function
Ferric chloride	0.2 g	Mordant
Cupric acetate	0.1 g	Mordant
Distilled water	100 mL	Solvent
Hydrochloric acid	2 mL	Acidifier

Compounding procedure

Make each solution seperately.
For use, combine equal parts of solutions A and B.
The working solution may be used immediately, but is not stable for long.

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for 5 seconds.
Rinse with tap water.
Dip briefly in 1% hydrochloric acid in 70% ethanol.
Wash well in running tap water to blue.
Rinse with distilled water.
Counterstain if desired.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – black
Background – as counterstain or unstained

Notes

The stock solutions are stable for some time.
The working solution should be made fresh.
This method is suitable as an acid resistant nuclear stain.
Only a very short staining time of 5 seconds is required.
The solution incorporates two mordants, iron and copper.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Faure, (1929)
Compte rendu hebdomadaire des séances et mémoires de la Société de biologie,
v.90, p.87.

March 30, 2023

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Cresyl Violet Stain for Nissl Bodies

By Carmen WongIntracytoplasmic Granules, Nissl Bodies, Protocols, Stain Target

Protocol

Cresyl Violet Stain

for Nissl Bodies

steps

materials

print

Materials

Staining Solution

Material	Amount
Cresyl violet	1	g
Distilled water	100	mL

Differentiator – Option 1

Material	Amount
Ethanol, 95%	100	mL
Cajeput oil	a few drops

Differentiator – Option 2

Material	Amount
Ethanol, 95%	100	mL
Acetic acid, glacial	5 drops

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives, particularly if ethanolic, are likely to be satisfactory.

Protocol

Bring sections to water via xylene and ethanol.
Place into the staining solution for 15-30 minutes.
Rinse with water, dehydrate with absolute ethanol and clear with xylene.
Leave in xylene for one hour.
Rinse well with absolute ethanol.
Bring one slide at a time to 95% ethanol.
Differentiate in either cajeput ethanol or acetic ethanol, controlling microscopically.
Rinse well with 95% ethanol to stop differentiation.
Dehydrate with absolute ethanol.
Clear with xylene
Coverslip using a resinous medium.

Expected Results

Nissl bodies – blue-violet
Nuclei – blue violet

Notes

The first dehydration and clearing of the stained section (steps 3-4) improves the sharpness of diffentiation and is recommended.
Differentiation, as in steps 5-10, may be repeated if necessary.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Drury, R A, and Wallington, E A, (1967).
Carleton’s histological technique., Ed. 5., pp. 379.
Oxford University Press, London, England.
Bench Manual

March 30, 2023

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Crossman’s Trichrome for Muscle and Collagen

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Crossman's Trichrome

for Muscle and Collagen

steps

materials

print

Materials

Solution A
Material Amount
Acid fuchsin 0.3 g
Orange G 2 g
Acetic acid, glacial 1 mL
Distilled water 100 mL
Thymol a small crystal
Solution B
Material Amount
Phosphomolybdic acid 1 g
Distilled water 100 mL
Solution C
- Option 1
  Material Amount
  Aniline blue 2 g
  Acetic acid, glacial 2 mL
  Distilled water 98 mL
- Option 2
  Material Amount
  Light green SF 1 g
  Acetic acid, glacial 1 mL
  Distilled water 99 mL
Solution D
Material Amount
Acetic acid, glacial 1 mL
Distilled water 99 mL

Material	Amount
Acid fuchsin	0.3	g
Orange G	2	g
Acetic acid, glacial	1	mL
Distilled water	100	mL
Thymol	a small crystal

Material	Amount
Phosphomolybdic acid	1	g
Distilled water	100	mL

Material	Amount
Aniline blue	2	g
Acetic acid, glacial	2	mL
Distilled water	98	mL

Material	Amount
Light green SF	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Material	Amount
Acetic acid, glacial	1	mL
Distilled water	99	mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation.

Protocol

Bring sections to water via xylene and ethanol.
Stain nuclei with an acid resistant nuclear stain such as Weigert’s iron hematoxylin.
Place into solution A for 1 minute.
Rinse with distilled water.
Place into solution B until collagen is decolorised.
Rinse quickly with distilled water.
Place into solution C for 5 minutes.
Rinse with distilled water.
Place into solution D until differentiated.
Rinse with distilled water.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – black
Erythrocytes – yellow
Cytoplasm – red
Collagen – blue or green

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.

March 30, 2023

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Csaba’s Stain for Mast Cells

By Carmen WongIntracytoplasmic Granules, Mast Cells, Protocols, Stain Target

Protocol

Csaba's Stain

for Mast Cells

steps

materials

print

Materials

Staining solution

Material	Amount
Alcian blue	0.36	g
Safranin	0.18	g
Ferric ammonium sulfate	0.48	g
Walpole’s acetate HCl buffer	100	mL

Walpole’s acetate-HCl buffer, pH 1.42

Material	Amount
Sodium acetate, M/1	100	mL
Hydrochloric acid, M/1	120	mL

Tissue Sample

Paraffin sections of neutral buffered formalin fixed tissue are suitable. Mercuric chloride fixatives are reputed to emphasise metachromasia. Other fixatives may be satisfactory.

Protocol

Bring sections to water via xylene and ethanol.
Place in to staining solution for 10-20 minutes.
Rinse with water.
Dehydrate with tertiary butanol.
clear with xylene and mount using a resinous medium.

Expected Results

Young mast cells – blue
Mature mast cells – red

Notes

To make an M/1 solution of sodium acetate, dissolve 82.04 grams anhydrous or 136.09 crystal sodium acetate in one litre of distilled water.
To make an M/1 solution of hydrochloric acid, dilute concentrated hydrochloric acid, which is about 10M, with distilled water (one part acid to 8 parts water) and titrate against an alkali of known molarity. Adjust the concentration to M/1 with distilled water.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Culling, C F A, (1974).
Handbook of Histopathological and Histochemical Techniques., Ed. 3. pp. 169 & 419
Butterworths, London, England.

March 30, 2023

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Papanicolaou’s Alcoholic Trichrome for Exfoliated Cells

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Papanicolaou's Alcoholic Trichrome

for Exfoliated Cells

steps

materials

print

This is the Papanicolaou technique commonly used to screen for cervical cancer.

Materials

Ehrlich’s or Harris’ alum hematoxylin or equivalent
Solution A (OG6)
Material Amount
Orange G 5 g
Phosphotungstic acid 0.15 g
Ethanol, 95% 1 L

Material	Amount
Orange G	5	g
Phosphotungstic acid	0.15	g
Ethanol, 95%	1	L

Solution B

Material	Variant
Material	EA 25		EA 36		EA 50		EA 65
Light green SF	2.2	g	2.25	g	0.45	g	1.125	g
Bismarck brown	0.6	g	0.5	g	0.5	g	0.5	g
Eosin Y	2.2	g	2.25	g	2.25	g	2.25	g
Phosphotungstic acid	1.7	g	2	g	2	g	2	g
Lithium carbonate	5	mg	5	mg	5	mg	5	mg
Ethanol, 95%	1	L	1	L	1	L	1	L

Preparation

For each variant:

Dissolve the bismarck brown in 100 mL ethanol, the light green in 450 mL ethanol, and the eosin Y in another 450 mL ethanol.
Combine the three solutions.
Add the acid and lithium carbonate.
Filter.

Tissue Sample

Ethanol-ether fixed smears of exfoliated epithelial cells.

Protocol

Bring smears to water via graded ethanols.
Stain nuclei with alum hematoxylin for 2 minutes.
Blue.
Rinse with 95% ethanol.
Place into solution A for 1 minute.
Rinse with 95% ethanol.
Place into solution B for 2 minutes.
Rinse well 95% ethanol.
Dehydrate rapidly with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Cells – shades of orange, pink or green

Notes

Although the method calls for smears fixed in an equal parts mixture of ethanol and diethyl ether, smears fixed in 95% or absolute ethanol are completely satisfactory.
Lithium carbonate saturates in water at 1% at room temperature, so 0.5 mL contains 5 mg.
EA 65 is recommended for specimens with large amounts of mucus.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Papanicolaou, (1942)
Science, v.95, pp.438
Culling C.F.A., (1974)
Handbook of histopathological and histochemical techniques Ed. 3
Butterworth, London, UK.
Bancroft, J.D. and Stevens A. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.

March 30, 2023

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