Friedlander's Alum Hematoxylin

steps

materials

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Materials

Material	Amount	Function
Hematoxylin	6 g	Dye
Potassium alum	6 g	Mordant
Distilled water	300 mL	Solvent
95% ethanol	300 mL	Solvent
Glycerol	300 mL	Stabiliser

Compounding procedures

Dissolve the Alum in the water.
Dissolve the hematoxylin in the ethanol.
Combine, and add glycerol.

Protocol

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

Expected Results

Nuclei – blue
Background – as counterstain or unstained

Notes

This is probably a regressive formula.
The staining time should be determined by trial.
Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

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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Shum & Hon’s PTAH

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

Protocol

Shum & Hon's PTAH

steps

materials

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Materials

Material	Amount	Function
Hematein,	0.8 g.	Dye
Phosphotungstic acid	9 g	Mordant
Distilled water	1 L	Solvent

Compounding procedures

Combine the hematein, phosphotungstic acid and 10 mL of the water and grind them to a paste with a pestle and mortar.
Wash the contents into a beaker with the rest of the water.
Bring the solution to a boil, then cool and filter.

Protocol

Bring sections to water with xylene and ethanol.
Perform a Mallory bleach using 0.25% potassium permanganate.
Rinse well with water.
Place into the PTAH solution for 12 – 24 hours at room temperature.
Rinse well with water.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Nuclei, erythrocytes, fresh fibrin, muscle striations – blue
Background – red

Notes

Observe the colour of the sludge while grinding as a good sample of hematein will be chocolate brown. If the sludge is pale, the final solution may not be satisfactory.
The staining may be done in an oven at about 60°C for a few hours, but the results are often less satisfactory.
Many substances have been stated to be stained blue. Sometimes the red staining overshadows blue staining, and the section may need to be washed to remove excess red.

Safety Note

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

References

Histological demonstration techniques, (1974)
Cook, H C.
Butterworths, London, England.

March 30, 2023

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Duval’s Alum Hematoxylin

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

Protocol

Duval's Alum Hematoxylin

steps

materials

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Materials

Original Formula

Material	Amount	Function
Hematoxylin, concentrated ethanolic	8 mL	Dye
Ammonium or potassium alum	a little	Mordant
Distilled water	800 mL	Solvent

Modern Formula

Material	Amount	Function
Hematoxylin, saturated ethanolic	8 mL	Dye
Ammonium or potassium alum	25 g	Mordant
Distilled water	800 mL	Solvent

Compounding procedure

Dissolve the alum in the water.
Add the hematoxylin.
The solution is likely progressive, although this is not stated to be so.

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for a few minutes.
Rinse with water and blue.
Rinse well with water.
Counterstain if desired.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Background – as counterstain or unstained

Notes

This solution is from the late 1800’s and is now obsolete, although the modern formula should stain satisfactorily.
Concentrated alcoholic hematoxylin, after ripening, would have contained no more than 7% hematein. It was made by soaking logwood chips in ethanol until no more dye dissolved out, then oxidized naturally. Depending on the sample of logwood and the amount of dye it contained, more than one batch may have been necessary to saturate the ethanol.
The type of alum was not specified, the most likely being either potassium or ammonium.
The formula calls for adding a “little alum” to 800 mL water. Potassium alum saturates at about 14% in water, so 800 mL would contain about 112 g. I have taken just less than 25% of the maximum (i.e. 25 grams) as being a “little”.
Of course, it could be any amount between 1 and 112 grams.
The appropriate time should be determined by trial. The instructions are to use full strength for a few minutes.
Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

Safety Note

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

References

Arthur Bolles-Lee, (1885)
The Microtomist’s Vade-Mecum
Originally published by: J & A Churchill, London, England.
Republished by: Science Heritage Ltd., Lincolnwood, Illinois, USA.
Susan Budavari, Editor, (1996)
The Merck Index, Ed. 12
Merck & Co., Inc., Whitehouse Station, NJ, USA

March 30, 2023

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Ehrlich’s Alum Hematoxylin

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

Protocol

Ehrlich's Alum Hematoxylin

steps

materials

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Materials

Material	Amount	Function
Hematoxylin	6 g	Dye
95% ethanol	300 mL	Solvent
Potassium alum	excess	Mordant
Distilled water	300 mL	Solvent
Glycerol	300 mL	Stabiliser
Glacial acetic acid	30 mL	Acidifier

Compounding procedures

Dissolve the hematoxylin in the ethanol mixed with acetic acid.
Dissolve the alum in the water mixed with glycerol in an oversized container.
Add the hematoxylin solution to the alum solution.
Plug the container loosely with cotton wool.
Ripen by leaving in a warm, sunlit place for several weeks.
When sufficiently ripened, store tightly stoppered in a cool, dark place.
The solution is stable for years.

Protocol

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

Expected Results

Nuclei – blue
Background – as counterstain or unstained

Notes

Gray gives 7 grams hematoxylin, and specifies ammonium alum.
The alum should be added to excess. This should be about 50 grams, but enough should be added to ensure undissolved alum is present.
This is a strongly staining, regressive formula. The staining time should be determined by trial. Usually, 20 minutes is adequate.
As with many strong alum hematoxylin solutions, cartilage, cement lines and mucin may stain blue.
The solution may be chemically ripened by adding 0.5g sodium iodate, but chemicallly ripened solutions are inferior in longevity.
Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

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.
Drury, R.A.B. and Wallington, E.A., (1980)
Carleton’s histological technique Ed. 5
Oxford University Press, Oxford, UK.
Bancroft, J.D. and Stevens A. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.
Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Ehrlich, (1886)
Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik, v. 3, p. 150.
Leipzig.

March 30, 2023

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Verhoeff’s Iron Hematoxylin for Elastic

By Carmen WongDye Type, Elastic Fibers, Hematoxylin and Eosin Staining, Iron Hematoxylin, Mordanted Hematoxylin, Protocols, Stain Target, Stain Type

Protocol

Verhoeff's Iron Hematoxylin

for Elastic

steps

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Materials

Stock Verhoeff A

Material	Amount
Hematoxylin	1	g
Ethanol, absolute	20	mL

Stock Verhoeff B

Material	Amount
Ferric chloride	10	g
Distilled water	100	mL

Stock Verhoeff C

Material	Amount
Potassium iodide	4	g
Iodine	2	g
Distilled water	100	mL

Working solution

Material	Amount
Stock solution A	20	mL
Stock solution B	8	mL
Stock solution C	8	mL

Differentiator

Material	Amount
Stock solution B	10	mL
Distilled water	40	mL

Tissue Sample

A 10% Formalin variant is suitable. Paraffin sections at 5µ are preferred.

Protocol

Bring sections to water via xylene and ethanol.
Place in working Verhoeff’s solution for 15 minutes.
Wash well with tap water to remove all excess hematoxylin.
Differentiate until the elastic fibres are satisfactory. This should be controlled microscopically based on the appearance of fibres in the area of interest.
Rinse well with tap water.
Place into 95% ethanol for 5 minutes to remove iodine discolouration.
Wash well with tap water to remove all residual chemicals.
Counterstain with Van Gieson.
Dehydrate with absolute ethanol.
Clear with xylene and mount with a resinous medium

Expected Results

Nuclei – black
Elastic fibres – black
Muscle – yellow
Collagen – red

Notes

The alcoholic hematoxylin solution should be freshly made. It does not need to have been ripened as ferric chloride is an oxidising agent. Some technologists keep pre-weighed aliquots of 1 gram hematoxylin and dissolve it in 20 mL ethanol as needed.
The working solution should be made just before use and allowed to stand for five minutes to ripen.
While this is a popular elastic stain, it is difficult to stain both coarse fibres and fine fibres optimally in a single section. If coarse fibres are well stained, the finest fibres may be completely decolourised, and if fine fibres are optimally stained, the coarse fibres are underdifferentiated. Ensure differentiation is optimised for the fibres of interest.
If Van Gieson is used as a counterstain, the elastic fibres should be very slightly underdifferentiated as picric acid will also remove some hematoxylin staining.
This method likely stains by two mechanisms: ionic attachment of iron mordanted hematoxylin to most structures and by van der Waal’s forces to elastic fibres. The differentiation step is accomplished either by oxidative bleaching of hematoxylin by ferric chloride, or by displacement of the mordanted hematoxylin by free mordant in solution. The dye attached to elastic fibres by van der Waal’s forces is not extracted easily. It is, however, still susceptible to bleaching, which likely explains why fine fibres may be pale. That bleaching takes place is shown by the differentiating fluid remaining clear during differentiation, extracted dye being seen to bleach as it dissolves out.
Note that nuclei are only adequately, not well, stained.

Safety Note

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

References

Histological demonstration techniques, (1974)
Cook, H C.
Butterworths, London, England
Culling, C.F.A., (1963)
Handbook of histopathological techniques, 2nd ed.
Butterworths, London.

March 30, 2023

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Delafield’s Alum Hematoxylin

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

Protocol

Delafield's Alum Hematoxylin

steps

materials

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Materials

Material	Amount	Function
Hematoxylin	6 g	Dye
Ammonium alum	90 g	Mordant
Distilled water	600 mL	Solvent
95% ethanol	200 mL	Solvent
Glycerol	150 mL	Stabiliser

Compounding procedures

Dissolve the hematoxylin in 50 mL ethanol.
Dissolve the alum in the water.
Add the two solutions.
Leave, loosely stoppered, in a warm, sunlit place for one week.
Filter, then add the glycerol and the rest of the ethanol.
Leave, loosely stoppered, in a warm, sunlit place for 3 months.
Filter and store in a tightly stoppered container in the dark.
This solution is stable for years.

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for 20 minutes.
Rinse well with water.
Differentiate with acid ethanol if necessary.
Rinse with water and blue.
Rinse well with water.
Counterstain if desired.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Background – as counterstain or unstained

Notes

The solution may be chemically ripened by adding 0.5 g sodium iodate, but such solutions are considered to be inferior in longevity.
Like many strong regressive formulations, cartilage, cement lines and mucins may be blue.
Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

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.
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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De Groot’s Alum Hematoxylin

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

Protocol

De Groot's Alum Hematoxylin

steps

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Materials

Material	Amount	Function
Hematoxylin	2 g	Dye
Ammonium alum	22 g	Mordant
Distilled water	270 mL	Solvent
95% ethanol	650 mL	Solvent
Glycerol	80 mL	Stabiliser
Hydrogen peroxide	7.5 mL	Oxidant
Potassium ferricyanide	0.8 g	–
Calcium chloride	15 g	–
Sodium bromide	7.5 g	–

Compounding procedures

Mix the ethanol, water and glycerol to make the solvent.
Add the peroxide to 15 mL of the solvent.
Add the hematoxylin, and dissolve.
Dissolve the calcium chloride and sodium bromide in 250 mL of the solvent.
Mix with the hematoxylin solution.
Add half the alum, and dissolve.
Dissolve the potassium ferricyanide in 400 mL of the solvent.
Add to the hematoxylin solution.
Dissolve the remaining alum in the remaining solvent.
Add to the hematoxylin solution.

Protocol

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

Expected Results

Nuclei – blue
Background – as counterstain or unstained

Notes

Both hydrogen peroxide and potassium ferricyanide are oxidizing agents. However, it is not clear if potassium ferricyanide is present for this reason.
The strength of hydrogen peroxide is not specified, but the commonest laboratory strength is 30 vols.
Although calcium can mordant hematoxylin, it is not clear if it is present for that reason.
The purpose of the sodium bromide is not clear. It may be present as a preservative, similar to chloral hydrate in some other formulas.
The staining time should be determined by trial.
Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

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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Debiden’s Alum Hematoxylin Variants

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

Protocol

Debiden's Alum Hematoxylin Variants

steps

materials

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Materials

Material	Variant		Function
Material	1987	1991	Function
Hematoxylin	5 g	5 g	Dye
Potassium alum	100 g	100 g	Mordant
Distilled water	1 L	1 L	Solvent
Glacial acetic acid	20 mL	20 mL	Acidifier
Mercuric oxide, red	2.5 g	–	Oxidant
Javex (5.25% sodium hypochlorite)	–	2 mL	Oxidant

Compounding procedures

Dissolve the alum in the water in a large flask.
Add the hematoxylin. Mix well.
Place in a 60-65°C oven overnight.
Remove from the oven, and add the appropriate oxidizing agent.
Stir until cold. Filter.

Preparation notes

1980

Acetic acid may be added as soon as it is cooled following preparation.
It is usable immediately.
It is preferable to prepare small volumes monthly.

1991

This improves in staining ability if allowed to ripen for a week or two.
If acetic acid is used, it should be added just before use.

Protocol

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

Expected Results

Nuclei – blue
Background – as counterstain or unstained

Notes

These two solutions are modifications of Harris’ hematoxylin and are regressive.
Paraffin sections should be stained for 5 minutes, and cytology smears for 45 seconds.
As with many strong regressive formulae, mucin may be stained blue.
Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

Safety Note

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

References

Debiden, D., (1987)
Improved preparation of Harris’ hematoxylin
Histologic, v.18, No.8
Debiden, D., (1991)
A new oxidant for Harris’ hematoxylin
Histologic, v.21, No.2

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

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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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Cook’s Copper Hematoxylin

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

Protocol

Cook’s Copper Hematoxylin

steps

materials

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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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