Nuclear Fast Red for Nuclei

By Carmen WongDye Type, Hematoxylin Alternatives, Protocols, Stain Type

Protocol

Nuclear Fast Red

for Nuclei

steps

materials

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Materials

Solution A

Material	Amount
Nuclear fast red	1	g
Potassium aluminum sulphate	50	g
Distilled water	500	mL

Preparation

Dissolve the dye and alum into the water.
Boil for 5 minutes. Cool and filter.

Solution B

Material	Amount
Tartrazine	1	g
Distilled water	1	L

Tissue Sample

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

Protocol

Perform the main staining method.
Place into solution A for 5 to 10 minutes
Rinse with water.
Optionally, place into solution B for 30 seconds.
Rinse with distilled water.
Return to the main staining method, or
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – red
Cytoplasm – yellow or unstained
Other tissues – according to the main staining method

Safety Note

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

References

Kiernan. J.A., (1999)
Histological and histochemical methods: Theory and practice, Ed. 3
Butterworth Heinemann, Oxford, UK.

March 30, 2023

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Pappenheim Stain for Plasma Cells

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

Protocol

Pappenheim Stain

for Plasma Cells

steps

materials

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Materials

Stock solution A

Material	Amount
Methyl green	1	g
Distilled water	100	mL
Phenol	0.25	g

Stock solution B

Material	Amount
Pyronin Y	4	g
Distilled water	100	mL
Phenol	5	g

Working solution

Material	Amount
Stock solution A	15	mL
Stock solution B	35	mL

Tissue Sample

Most fixatives should be satisfactory.

Protocol

Bring sections to water via xylene and ethanol.
Place into the staining solution for 5-10 minutes.
Rinse briefly with water.
Dehydrate rapidly with acetone.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – violet
Plasma cell cytoplasm – red
Other cell cytoplasm – pink

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. p. 355
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.

March 30, 2023

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Sandifords Stain for Plasma Cells

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

Protocol

Sandifords Stain

for Plasma Cells

steps

materials

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Materials

Staining solution

Material	Amount
Distilled water	75	mL
Glycerol	20	mL
Ethanol, 95%	5	mL
Methyl green	0.15	g
Pyronin Y	0.5	g
Phenol	1.5	g

Tissue Sample

Most fixatives should be satisfactory if fixation is not extended. Reagents which cause depolymerisation of DNA should be avoided. Decalcification may interfere with staining.

Protocol

Bring sections to water via xylene and ethanol.
Place into the staining solution for 5-10 minutes.
Rinse briefly with water.
Dehydrate rapidly with acetone.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – violet
Plasma cell cytoplasm – red
Other cell cytoplasm – pink

Notes

The reference gave no details of the method to use. The details above are from that given for Pappenheim’s solution, and should be suitable as a starting point.
Time and temperature of staining may need to be increased.

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. p. 355
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.

March 30, 2023

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Unna’s Stain for Plasma Cells

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

Protocol

Unna's Stain

for Plasma Cells

steps

materials

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Materials

Staining Solution

Material	Amount
Distilled water	100	mL
Phenol	0.5	g
Glycerol	20	mL
Ethanol, absolute	2.5	mL
Pyronin Y	0.25	g
Methyl green	0.15	g

Preparation

Place both dyes in a mortar, add the ethanol and grind together.
Heat the glycerol to 50°C and add small volumes to the dyes while grinding.
Dissolve the phenol in the water and wash the dyes from the mortar. Filter.

Tissue Sample

Most fixatives should be satisfactory.

Protocol

Bring sections to water via xylene and ethanol.
Place into the staining solution at 30°C for 10 minutes.
Rinse briefly with water.
Place in absolute ethanol until differentiated.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – violet
Plasma cell cytoplasm – red
Other cell cytoplasm – pink

Notes

In their description of this method, Carleton and Leach omit the method of preparation, implying that the trituration of the dyes may not be necessary.

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. p. 355
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Carleton, H M, and Leach, E H, (1938).
Histological technique., Ed. 2.
Oxford University Press, London, England.

March 30, 2023

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Papamiltiades’ Alum Hematoxylin

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

Protocol

Papamiltiades' Alum Hematoxylin

steps

materials

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Materials

Material	Amount	Function
Hematoxylin	4 g	Dye
Aluminum sulphate	10 g	Mordant
Distilled water	900 mL	Solvent
Zinc sulphate	5 g	–
Potassium iodide	4 g	Stabiliser
Glacial acetic acid	32 mL	Acidifier
Glycerol	100 mL	Stabiliser

Compounding Procedure

Dissolve the hematoxylin in 400 mL water.
Dissolve the aluminum sulphate in 200 mL water.
Dissolve the zinc sulphate in 100 mL water.
Dissolve the potassium iodide in 100 mL water.
Combine the four solutions, then add the acetic acid and glycerol.
The solution may be used immediately, and is stable for about two months.

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for an appropriate time.
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 is progressive.
The purpose of the zinc sulphate and potassium iodide are not clear.
The staining time should be determined by trial.
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

Putt, F.A.
Manual of Histopathological Staining Methods
John Wiley & Sons, New York, NY., USA

March 30, 2023

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Paquin & Goddard’s Iron Hematoxylin

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

Protocol

Paquin & Goddard's Iron Hematoxylin

steps

materials

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Materials

Hematoxylin Solution

Material	Amount	Function
Hematoxylin	0.8 g	Dye
Ferric ammonium sulfate	5 g	Mordant
Ammonium sulfate	0.7 g	–
95% ethanol	25 mL	Solvent
Glycerol	13 mL	Solvent
Distilled water	75 mL	Solvent

Picric Ethanol

Material	Amount	Function
Picric acid, saturated ethanolic.	6 mL	Acid
95% ethanol	94 mL	Solvent

Compounding Procedure

Combine the glycerol and ethanol.
Add the hematoxylin and dissolve using gentle heat.
Dissolve the other ingredients in the water.
Add slowly to the hematoxylin solution with agitation.
Let stand for 24 hours before use.

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for 5 minutes.
Rinse with tap water.
Differentiate briefly in picric 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

This was specified as an acid resistant nuclear stain prior to Paquin & Goddard’s trichrome. It is also suitable for other methods requiring an acid resistant nuclear stain.

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:
Paquin and Goddard, (1947)
Bulletin of the International Association of Medical Museums
and Journal of Technical Methods, v. 27, p. 198

March 30, 2023

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Phenolic MGP Solutions

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

Protocol

Phenolic MGP Solutions

steps

materials

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Materials

Table 1. Comparison of phenolic methyl green –pyronin solutions for plasma cells

Material	Formula
Material	Pappenheim		Unna		Sandiford		Scott & French		Langeron		Kurnick
Distilled water	100	mL	82	mL	75	mL	80	mL	100	mL	100	mL
Glycerol	–	mL	16	mL	20	mL	16	mL	–	mL	–	mL
Ethanol, 100%	0	mL	2	mL	0	mL	4	mL	–	mL	–	mL
Ethanol, 95%	–	mL	–	mL	5	mL	–	mL	–	mL	–	mL
Phenol	0.25	g	0.4	g	1.5	g	1.6	g	5	g	–	g
Methyl green	0.3	g	0.12	g	0.15	g	0.8	g	2	g	0.6	g
Pyronin Y	0.7	g	0.2	g	0.5	g	0.2	g	2	g	1	g

Tissue Sample

Most fixatives should be satisfactory.

Protocol

These solutions have been included for comparison purposes, and the formulae have been adjusted to make 100 mL of each solution to facilitate the comparison. See the individual methods for details of their use and the actual formula. There is no single method for their application, but the details below may be used as a starting point.

Bring sections to water via xylene and ethanol.
Place in the MGP solution for 10 minutes at room temperature.
Rinse with distilled water.
Differentiate with absolute ethanol if required.
Complete dehydration with acetone if necessary.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue-green
Plasma cell cytoplasm – red
Other cell cytoplasm – pink

Notes

The time and temperature in step 2 may need to be increased.
Differentiation may not be required, depending on the time and temperature of staining.
Various fluids have been used for dehydration: acetone, n-butanol, t-butanol, 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. p. 355
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Disbrey, B. D., (1970)
Histological laboratory methods.
E. & S. Livingstone, Edinburgh and London, UK.

March 30, 2023

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Pollak’s Trichrome for Muscle and Collagen

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

Protocol

Pollak's Trichrome

for Muscle and Collagen

steps

materials

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Materials

Weigert’s iron hematoxylin or equivalent
Solution A
Material Amount
Orange G 0.25 g
Ponceau 2R 0.33 g
Acid fuchsin 0.17 g
Light green SF 0.15 g
Phosphotungstic acid 0.5 g
Phosphomolybdic acid 0.5 g
Acetic acid, glacial 1 mL
Ethanol, 95% 50 mL
Distilled water 50 mL
Solution B
Material Amount
Acetic acid, glacial 0.2 mL
Distilled water 100 mL

Material	Amount
Orange G	0.25	g
Ponceau 2R	0.33	g
Acid fuchsin	0.17	g
Light green SF	0.15	g
Phosphotungstic acid	0.5	g
Phosphomolybdic acid	0.5	g
Acetic acid, glacial	1	mL
Ethanol, 95%	50	mL
Distilled water	50	mL

Material	Amount
Acetic acid, glacial	0.2	mL
Distilled water	100	mL

Preparation of Solution A

Mix the water, ethanol and acetic acid together.
Divide into four, and dissolve as follows:
1. Phosphomolybdic acid
2. Phosphotungstic acid and orange G
3. Light green SF
4. Ponceau 2R and acid fuchsin
Combine all solutions and filter.

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. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

Protocol

Bring sections to water via xylene and ethanol.
Stain nuclei with Weigert’s iron hematoxylin or equivalent.
Place into solution A for 3-7 minute.
Place into solution B until differentiated.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – black
Erythrocytes – yellow
Cytoplasm – red
Muscle – red
Collagen – blue

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:
Pollack, (1944)
Archives of pathology and laboratory medicine, v. 37, pp. 294
Chicago, USA

March 30, 2023

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Pusey’s Alum Hematoxylin

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

Protocol

Pusey's Alum Hematoxylin

steps

materials

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This solution is described as a modification of Mayer’s hemalum, but please refer to that formula.

Materials

Material	Amount	Function
Hematoxylin, aged 10% ethanolic	16 mL	Dye
Ammonium alum	60 g	Mordant
Distilled water	1 L	Solvent
Sodium iodate	0.25 g	Oxidant
Citric acid, 5% aqueous	7 mL	Acidifier
Chloral hydrate	50 g	Stabiliser

Compounding Procedure

Dissolve the Alum in the water using low heat, but do not boil.
Add the hematoxylin solution and mix well.
Add the sodium iodate and dissolve.
Leave for 30 minutes.
Add the chloral hydrate and dissolve.
Add the citric acid and mix well.
Check that the pH is 2.45, adjust with citric acid if not.
It may be used immediately.

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for 5-10 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

The alcoholic hematoxylin solution should be aged (partially oxidised).
The staining time was not given, but 5-10 minutes should suffice.
16 mL of a 10% solution of hematoxylin equates to 1.6 grams dye. With 60 grams of mordant and strong oxidation this would indicate a darkly staining progressive solution.
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

Pusey’s modified Mayer’s hematoxylin,
Journal of Histotechnology, v.2, No.2, p.54, 1979
citing:
Villaneuva, A.R., (1976)
Methods of preparing and interpreting mineralized sections of bone, in:
Proceedings of the First Workshop on Bone Morphometry
Jaworski, Z. F. G., Editor, (1976).

March 30, 2023

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Rawitz’ Alum Hematoxylin Variants

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

Protocol

Rawitz' Alum Hematoxylin Variants

steps

materials

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Materials

Material	Variant			Function
Material	1895a	1895b	1909	Function
Hematoxylin	10 g	–	–	Dye
Hematein	–	2.5 g	2 g	Dye
Potassium alum	10 g	–	–	Mordant
Ammonium alum	–	15 g	–	Mordant
Aluminum nitrate	–	–	20 g	Mordant
Distilled water	650 mL	500 mL	500 mL	Solvent
Glycerol	350 mL	500 mL	500 mL	Stabiliser

Compounding Procedure

Dissolve the aluminum salt and dye in the water.
When dissolved, add glycerol.
The 1895a formula should be allowed to ripen.
The 1895b and 1909 formulas may be used immediately.

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

The 1895b and 1909 formulae use hematein instead of hematoxylin.
The Microtomists Formulary and Guide gives the 1895b and 1909 formulae as containing 500 millilitres of glycerol. The Microtomists Vade-Mecum gives these two formulae as containing 500 grams of glycerol. This equates to 400 millilitres based on a specific gravity of 1.25.
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.
Bolles Lee, A.. Edited by Gatenby, J.B. and Beams, H.W., (1950)
The Microtomist’s Vade-Mecum. 11 ed.,
Churchill, London, UK.

March 30, 2023

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