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Dye Type

Papamiltiades’ Alum Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Papamiltiades' Alum Hematoxylin

6
steps
7
materials
print

Materials

MaterialAmountFunction
Hematoxylin4 gDye
Aluminum sulphate10 gMordant
Distilled water900 mLSolvent
Zinc sulphate5 g–
Potassium iodide4 gStabiliser
Glacial acetic acid32 mLAcidifier
Glycerol100 mLStabiliser

Compounding Procedure

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

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse with water and blue.
  4. Rinse well with water.
  5. Counterstain if desired.
  6. 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

  1. 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 Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Paquin & Goddard's Iron Hematoxylin

8
steps
7
materials
print

Materials

Hematoxylin Solution

MaterialAmountFunction
Hematoxylin0.8 gDye
Ferric ammonium sulfate5 gMordant
Ammonium sulfate0.7 g–
95% ethanol25 mLSolvent
Glycerol13 mLSolvent
Distilled water75 mLSolvent

Picric Ethanol

MaterialAmountFunction
Picric acid, saturated ethanolic.6 mLAcid
95% ethanol94 mLSolvent

Compounding Procedure

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

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 5 minutes.
  3. Rinse with tap water.
  4. Differentiate briefly in picric ethanol.
  5. Wash well in running tap water to blue.
  6. Rinse with distilled water.
  7. Counterstain if desired.
  8. 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

  1. 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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Pusey’s Alum Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Pusey's Alum Hematoxylin

6
steps
6
materials
print

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

Materials

MaterialAmountFunction
Hematoxylin, aged 10% ethanolic16 mLDye
Ammonium alum60 gMordant
Distilled water1 LSolvent
Sodium iodate0.25 gOxidant
Citric acid, 5% aqueous7 mLAcidifier
Chloral hydrate50 gStabiliser

Compounding Procedure

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

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 5-10 minutes.
  3. Rinse with water and blue.
  4. Rinse well with water.
  5. Counterstain if desired.
  6. 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

  1. 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 Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Rawitz' Alum Hematoxylin Variants

8
steps
7
materials
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Materials

MaterialVariantFunction
1895a1895b1909
Hematoxylin10 g––Dye
Hematein–2.5 g2 gDye
Potassium alum10 g––Mordant
Ammonium alum–15 g–Mordant
Aluminum nitrate––20 gMordant
Distilled water650 mL500 mL500 mLSolvent
Glycerol350 mL500 mL500 mLStabiliser

Compounding Procedure

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

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse well with water.
  4. Differentiate with acid ethanol if necessary.
  5. Rinse with water and blue.
  6. Rinse well with water.
  7. Counterstain if desired.
  8. 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

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

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Régaud's Iron Hematoxylin

9
steps
7
materials
print

Materials

Solution A

MaterialAmountFunction
Ferric ammonium sulfate5 gMordant
Distilled water100 mLSolvent

Solution B

MaterialAmountFunction
Hematoxylin1 gDye
90% ethanol10 mLSolvent
Distilled water80 mLSolvent
Glycerol10 mLStabiliser

Solution C

MaterialAmountFunction
Potassium ferricyanide2.5 gBleach
Sodium borate2 gpH control
Distilled water100 mLSolvent

Compounding Procedure

  1. Make each solution separately.
  2. Solutions A and B may be reused provided they are not contaminated by each other.
  3. Solution C should not be reused.

Protocol

  1. Bring sections to distilled water with xylene and ethanol.
  2. Place into solution A at 50°C for 30 minutes.
  3. Rinse well with distilled water.
  4. Place into solution B at 50°C for 30 minutes.
  5. Rinse well with distilled water.
  6. Place into solution C until differentiated.
  7. Wash in running tap water until blue.
  8. Counterstain as required.
  9. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Background  –  as counterstain or unstained

Notes

  • The solutions are stable for some time.
  • Solution C is Weigert’s ferricyanide bleach and should be used fresh.

Safety Note

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

References

  1. 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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Rozas’ Iron Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Rozas' Iron Hematoxylin

7
steps
8
materials
print

Rozas’ iron hematoxylin is of interest as it contains aluminum and iron salts in one solution. Both are mordants used with hematoxylin, and there will presumably be some kind of competition for available binding sites which could modify the solution’s staining characteristics. Since iron can be used to displace aluminum as a mordant for hematoxylin, it is likely that the solution stains more as an iron hematoxylin than as an aluminum hematoxylin.

Materials

Solution A

MaterialAmountFunction
Hematoxylin0.6 gDye
Ferric ammonium sulfate1 gMordant
Aluminum chloride1.2 gMordant
Distilled water74 mLSolvent
95% ethanol6 mLSolvent
Glycerol20 mLStabiliser

Solution B

MaterialAmountFunction
Ferric ammonium sulfate20 gMordant
Distilled water100 mLSolvent

Compounding Procedure

  1. For solution A, dissolve the hematoxylin in the ethanol.
  2. Dissolve the ferric ammonium sulfate and the aluminum chloride in the water.
  3. Combine, then add the glycerol.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into solution A for 12-24 hours.
  3. Place into solution B until differentiated.
  4. Wash well in running tap water to blue.
  5. Rinse with distilled water.
  6. Counterstain if desired.
  7. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Background  –  as counterstain or unstained

Notes

  • The presence of glycerol indicates that solution A may be stable for some time.
  • The staining procedure indicates that the technique is intended as a primary stain. It is likely a modification of Heidenhain’s method and would demonstrate much the same material.

Safety Note

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

References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    Rozas, (1935)
    Zietschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik,
    v.52, p.1. Leipzig.
March 30, 2023
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Nuclear Fast Red for Nuclei

By Dye Type, Hematoxylin Alternatives, Protocols, Stain Type
Protocol

Nuclear Fast Red

for Nuclei

7
steps
5
materials
print

Materials

Solution A

MaterialAmount
Nuclear fast red1g
Potassium aluminum sulphate50g
Distilled water500mL

Preparation

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

Solution B

MaterialAmount
Tartrazine1g
Distilled water1L

Tissue Sample

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

Protocol

  1. Perform the main staining method.
  2. Place into solution A for 5 to 10 minutes
  3. Rinse with water.
  4. Optionally, place into solution B for 30 seconds.
  5. Rinse with distilled water.
  6. Return to the main staining method, or
  7. 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

  1. Kiernan. J.A., (1999)
    Histological and histochemical methods: Theory and practice, Ed. 3
    Butterworth Heinemann, Oxford, UK.
March 30, 2023
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Mallory’s Alum Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Mallory's Alum Hematoxylin

8
steps
4
materials
print

Materials

MaterialAmountFunction
Hematoxylin2.5 gDye
Potassium alum50 gMordant
Distilled water1 LSolvent
Thymol2.5 gPreservative

Compounding Procedure

  1. Dissolve the dye and Alum in the water.
  2. Add the thymol.
  3. Allow to ripen before use.

    4. </ol

Protocol

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

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • 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

  1. 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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Mallory & Wright’s Alum Hematoxylin Variants

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Mallory & Wright's Alum Hematoxylin Variants

8
steps
3
materials
print

Materials

MaterialStandard FormulaStrong FormulaFunction
Hematoxylin1 g1 gDye
Ammonium alum, saturated aqueous100 mL100 mLMordant
Distilled water300 mL–Solvent

Compounding Procedure

  1. Dissolve the hematoxylin in the Alum solution by warming if necessary.
  2. Add the water if the standard solution is being used.
  3. Plug the container with cotton wool.
  4. Ripen for approximately ten days.
  5. Place the stopper tightly.
  6. Filter before use. The solution is stable for 2-3 months.
  7. A small crystal of thymol may be added.

Protocol

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

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • The standard solution is for routine, formalin fixed tissues.
  • The strong solution is recommended for Zenker fixed tissues.
  • 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

  1. Mallory, F. B. & Wright, J.H., (1904)
    Pathological technique, Ed.3
    W. B. Saunders, Philadelphia, USA.
March 30, 2023
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Mallory’s PTAH

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Mallory's PTAH

6
steps
3
materials
print

Materials

MaterialAmountFunction
Hematein,1 g.Dye
Phosphotungstic acid10 gMordant
Distilled water1 LSolvent

Compounding Procedure

  1. Dissolve the hematein in 200 mL of the water and the phosphotungstic acid in the rest.
  2. Combine the solutions.
  3. Keep in a bottle with a tightly fitting cap to ensure atmospheric oxygen is excluded.
  4. The stain may be used after a day or two.

Protocol

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

Expected Results

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

Notes

  • If formalin fixed, the sections may be treated with acid dichromate for 30 minutes.
    MaterialAmount
    Potassium dichromate, 3% aqueous36mL
    10% hydrochloric acid in absolute ethanol.12mL
  • 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 with water to remove excess red. Excess blue may be removed by extending the treatment with ethanol during dehydration.
  • PTAH may also be made with hematoxylin and oxidized as any other hematoxylin solution. Atmospheric oxidation is often recommended, but takes a few months. Place the solution in a flask with a loose cotton batting stopper to facilitate exposure to air. Test periodically and, when staining is satisfactory, place into a bottle with a tightly fitting lid to inhibit atmospheric oxidation.
  • Alternatively, the hematoxylin may be chemically oxidized. This is usually done with 12 mL of 1% aqueous potassium permanganate. The solution may be used after a day or two. Sodium iodate may also be used. Half the hematoxylin will be oxidized with 0.1 grams sodium iodate. If brought to a boil and allowed to cool, it may be used immediately, or it may be allowed to oxidize at room temperature for a few days.

Safety Note

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

References

  1. Bancroft, J. D. and Stevens, A.,
    Theory and Practice of Histological Techniques, Ed. 2,
    Churchill Livingstone, London, UK.
March 30, 2023
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