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Mordanted Hematoxylin

Weigert’s Iron Hematoxylin

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

Weigert's Iron Hematoxylin

8
steps
8
materials
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Materials

Solution A

MaterialVariantFunction
19031904
Ferric chloride0.4 g0.6 gMordant
Distilled water100 mL100 mLSolvent
Hydrochloric acid–0.75 mLSolvent

Solution B

MaterialVariantFunction
19031904
Hematoxylin1 g1 gDye
95% ethanol100 mL100 mLSolvent

Solution C

MaterialVariantFunction
19031904
Potassium ferricyanide2.5 g–Bleach
Sodium borate2 g–Alkaliniser
Distilled water100 mL–Solvent

Note: The 1904 formula is the solution usually meant when Weigert’s iron hematoxylin is specified as an acid resistant nuclear stain.

Compounding Procedure

For both variants:

  1. Make each solution separately.
  2. Filter.
  3. Immediately before use, combine equal parts of solutions A and B.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 1-2 hours.
  3. Rinse with tap water.
  4. 1903 – Place in solution C until differentiated.
    1904 – Dip briefly in 1% hydrochloric acid in 70% 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

  • The stock solutions are stable for some time.
  • The working solution should be made fresh.
  • The alcoholic hematoxylin solution was originally made by diluting a stock solution of 10% hematoxylin in 95% ethanol.
  • Solution A of the 1904 variant was originally made from a commercial ferric chloride solution, the amounts above being given by Gray based on its formula. The following is now usually specified:30% aqueous ferric chloride – 4 mL

    Distilled water – 100 mL

    Hydrochloric acid – 1 mL

  • Both methods were originally intended to be used without a counterstain for demonstrating chromatin and other structures usually stained by iron hematoxylin. If the 1904 formula is used for this purpose, the staining time should be increased and the excess stain removed with acid ethanol.

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:–
    Ehrlich, P., Krause, R. et. al., (1910)
    Enzyklopädie der mikroskopischen technik, ed. 2, v. 1, p. 231.
    Weigert, K., (1904)
    Zeitschrift für wissenschaftliche mikroskopie und für mikroskopische technik,
    v. 21, p. 1.
  2. 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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Yasvoyn’s Iron Hematoxylin

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

Yasvoyn's Iron Hematoxylin

5
steps
3
materials
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Materials

Solution A

MaterialAmountFunction
Hematoxylin0.1 gDye
Distilled watermLSolvent

Solution B

MaterialAmountFunction
Ferric ammonium sulfate2.5 gMordant
Distilled water100 mLSolvent

Compounding Procedure

  1. Make each solution separately.
  2. For use, add solution B drop by drop to 20 drops of solution A until it just remains blue .
  3. The working solution may be used immediately, but is not stable for long.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 2-5 minutes.
  3. Rinse with water.
  4. Remove excess stain with 70% ethanol if necessary.
  5. 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.
  • Although not specified, a tap water wash to blue the hematoxylin may be useful following step 4.
  • Following a wash to blue the nuclei, a counterstain could probably be applied, if wished, before dehydration.

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:
    Roskin, G.E, (1946).
    Mikroskopecheskaya technica, p. 150
March 30, 2023
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Thomas’ Iron Hematoxylin

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

Thomas' Iron Hematoxylin

6
steps
8
materials
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Materials

Solution A

MaterialAmountFunction
Hematoxylin2.5 gDye
Dioxane40 mLSolvent
Hydrogen peroxide1 mLoxidizer

Solution B

MaterialAmountFunction
Ferric ammonium sulfate3 gMordant
Ferric chloride1.5 gMordant
Ferrous chloride6 gMordant
Distilled water60 mLSolvent
Acetic acid6 mLAcidifier

Compounding Procedure

  1. Dissolve the hematoxylin into the dioxane.
  2. Add the peroxide.
  3. Add the acetic acid to the water.
  4. Dissolve the iron salts into the dilute acetic acid.
  5. Filter the solution of iron salts into the hematoxylin solution.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Wash well in running tap water to blue.
  4. Rinse with distilled water.
  5. Counterstain if desired.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Background  –  as counterstain or unstained

Notes

  • The solution is likely stable for some time.
  • Dioxane is no longer used in histotechnology for safety reasons.The method should be considered obsolete.
  • The solution is progressive, and the staining time should be determined by trial.

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:
    Thomas, (1943)
    Bulletin d’histologie appliquée à la physiologie et à la pathologie et de technique microscopique, v. 20, p. 212.
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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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
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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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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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Molnar’s Alum Hematoxylin Variants

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

Molnar's Alum Hematoxylin Variants

8
steps
9
materials
print

Materials

MaterialVariation IVariation IFunction
Hematoxylin10 g4 gDye
Ammonium or potassium alum50 g50 gMordant
Distilled water1 L1 LSolvent
95% ethanol50 mLSolvent
Mercuric oxide5 gOxidant
Sodium iodate0.3 gOxidant
Glacial acetic acid20 mLAcidifier
Citric acid1.5 gAcidifier
Chloral hydrate75 gAntioxidant

Compounding Procedures

Variation I

  1. Dissolve the dye into the ethanol, and the Alum into the water.
  2. Combine the solutions in an Erlenmeyer flask and slowly add the mercuric oxide
  3. Reheat until the solution changes colour to a dark purple.
  4. Remove from heat and cool rapidly by swirling the flask in cold water.
  5. When cool, add the acetic acid.

Variation II

  1. Dissolve the Alum into the water.
  2. Add the hematoxylin, sodium iodate, citric acid and chloral hydrate in that order.
  3. Filter before use.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for the specified time.
    1. Var I for 10 minutes.
    2. Var II for 8-10 seconds.
  3. Rinse well with water.
  4. Differentiate Var I with acid ethanol for a few seconds.
  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

  • Var I was recommended for double embedded tissues.
  • The use of mercuric oxide as an oxidant is now deprecated due to its toxicity. Var I could be modified by using sodium iodate at about 0.3 grams instead.
  • 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. Molnar, L. N.,
    Modification of Harris hematoxylin for sections from tissue double embedded with nitrocellulose and paraffin.
    Histologic, v 5, Nº 1, January, 1975
  2. Molnar, L. N.,
    Modification of Mayer’s hematoxylin-eosin method.
    Histologic, v 6, Nº 4, October, 1976
March 30, 2023
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