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Mordanted Hematoxylin

Roach & Smith’s Bismuth Hematoxylin

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

Roach & Smith's Bismuth Hematoxylin

4
steps
7
materials
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This method demonstrates arginine.

Materials

MaterialAmountFunction
Hematoxylin50 mgDye
Ethanol, abs.5 mLSolvent
Sodium iodate10 mgOxidant
Bismuth nitrate50 mgMordant
Citric acid, 0.5M aqu.85 mLAcidifier
Glycerol20 mLSolvent
Sodium hydroxide, 1 M aqu.19 mLAlkaliser

Compounding procedure

  1. Dissolve the bismuth nitrate into the citric acid solution.
  2. Add 10 mL of the glycerol, then the sodium hydroxide solution.
  3. Adjust the pH to 5.2.
  4. Dissolve the hematoxylin into the ethanol.
  5. Combine the two solutions.
  6. Add the sodium iodate dissolved in 1 mL distilled water.
  7. Stir for 20 minutes.
  8. Add the remaining 10 mL of glycerol.

This solution has a usable life of about 3 hours.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into bismuth hematoxylin for 10 minutes.
  3. Rinse with water.
  4. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Arginine residues  –  black

Notes

  • The formula calls for bismuth nitrate pentahydrate, Bi(NO3)3.5H20.
  • This method demonstrates proteins with an arginine content of about 12% or more.
  • Nuclear histones and myelin basic protein stain strongly.
  • The authors suggest that the bismuth binds to the guanidine groups of arginine.

Safety Note

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

References

  1. Roach, J. B., and Allen, A. A., (1997)
    Bismuth hematoxylin stain for arginine residues.
    Biotechnic & Histochemistry, v.72, Nº 1, p. 49.
March 30, 2023
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Apathy’s Alum Hematoxylin

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

Apathy's Alum Hematoxylin

8
steps
7
materials
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Materials

MaterialAmountFunction
Hematoxylin3 gDye
Ammonium alum30 gMordant
Distilled water450 mLSolvent
95% ethanol250 mLDye solvent
Glycerol350 mLStabiliser
Glacial acetic acid10 mLAcidifier
Salicylic acid0.3 gAcidifier

Compounding procedure

  1. Dissolve the dye in 100 mL water and 250 mL ethanol.
  2. Leave at room temperature to ripen (months).
  3. Dissolve the alum and acids in 350 mL water.
  4. Combine both solutions.
  5. Add glycerol.

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

  • Lillie excludes the salicylic acid.
  • The appropriate 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. Lillie, R.D., (1954)
    Histopathologic technique and practical histochemistry Ed.2
    Blakiston, New York, USA.
March 30, 2023
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Anderson’s Iron Hematoxylin

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

Anderson's Iron Hematoxylin

6
steps
7
materials
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Materials

Solution A

MaterialAmountFunction
Hematoxylin0.5 gDye
100% ethanol50 mLSolvent
Distilled water50 mLSolvent
Calcium hypochlorite, 2% aqueous5 mLOxidant

Solution B

MaterialAmountFunction
Ferric ammonium sulphate3 gMordant
Distilled water100 mLSolvent
Sulfuric acid0.5 mLAcidifier

Compounding procedures

  1. Make each solution separately.
  2. For use, add 2 volumes of solution A to 1 volume of solution B.
  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 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 stock solutions are stable for some time.
  • The working solution should be made fresh.
  • It is claimed that the working solution rarely overstains, i.e. it is progressive.
  • Solution A incorporates calcium hypochlorite as an oxidizing agent for hematoxylin. Presumably other oxidizing agents would suffice equally as well. Sodium iodate (0.1 g or less) is the most common.
  • 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:
    Anderson, J., (1929)
    How to stain the nervous system.
    Livingstone. Edinburgh, Scotland.
March 30, 2023
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Anderson’s Alum Hematoxylin

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

Anderson's Alum Hematoxylin

8
steps
7
materials
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Although there are three variations listed for Anderson’s formulas, those marked as 1923a and 1923b are variations of the same solution, the former being that given in the Microtomist’s Formulary and Guide and the latter in the Microtomist’s Vade Mecum.

Materials

MaterialVariantFunction
1923a1923b1929
Hematoxylin2.5 g2.5 g5 gDye
Ammonium alum20 gSat.30 gMordant
Distilled water900 mL900 mL700 mLSolvent
95% ethanol50 mL50 mL50 mLSolvent
Calcium hypochlorite4 g–40 gOxidant
Chloramine T or lime chloride4 gOxidant
Glacial acetic acid50 mL50 mL50 mLAcidifier

Compounding procedures

1923a & 1929

  1. Dissolve the calcium hypochlorite in 200 mL water.
  2. Dissolve the hematoxylin in some of the water.
  3. After 4 hours, combine the solutions.
  4. Dissolve the other ingredients in the rest of the water.
  5. Combine with the hematoxylin-hypochlorite solution.

1923b

  1. Bring 700 mL water to a boil, then saturate it with alum (see notes).
  2. Allow to cool for one day, then filter.
  3. Dissolve the chloramine T or lime chloride into 200 mL of water.
  4. Leave four hours. Shake occasionally.
  5. Dissolve the hematoxylin in the ethanol
  6. Add the oxidant solution to the hematoxylin.
  7. Mix for a few seconds. It should be dark brown.
  8. Slowly add to the 700 mL alum solution, while shaking.
  9. Add the acetic acid.
  10. It is ready immediately.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 2-3 minutes.
  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 1923a formula oxidizes 2.5 grams hematoxylin with 4 grams calcium hypochlorite. This is a ratio of 1.6:1.
  • The 1929 formula oxidizes 5 grams hematoxylin with 40 grams calcium hypochlorite. This is a ratio of 8:1.
  • The 1923b formula is an alternate formula given by Bolles-Lee. It differs from the 1923a formula by using a saturated alum solution, and chloramine T or “commercial chloride of lime” (a crude preparation of calcium chloride with other substances present) as oxidant.
  • The 1923b formula calls for 700 mL saturated aqueous alum. The instructions specify that the water should be saturated at boiling, then cooled to room temperature. This would require about 350 grams alum (at 500 mg/mL), but at room temperature, the solution would contain only about 100 grams (at 150 mg/mL). Dissolving 110 grams alum in 700 mL hot water, cooling and filtering would give the same solution.
  • 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.
    Citing:
    Anderson, J., (1923)
    Journal of Pathology and Bacteriology.
  3. Susan Budavari, Editor, (1996)
    The Merck Index, Ed. 12
    Merck & Co., Inc., Whitehouse Station, NJ, USA
March 30, 2023
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Akita & Kaneko’s Hemalum for Elastic Fibers

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

Akita & Kaneko's Hemalum

for Elastic Fibers

5
steps
6
materials
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Materials

MaterialAmount
Hematoxylin100mg
Ethanol, 70%100mL
Sodium iodate20mg
Potassium alum300mg
Chloral hydrate5g
Citric acid100mg

Staining Solution Preparation

  1. Dissolve the hematoxylin into the ethanol.
  2. Add the potassium alum, then the other ingredients.
  3. Mix well for 15 to 20 minutes, then filter.
  4. At this point the solution is stable for 2 months at room temperature.
  5. Immediately prior to use, adjust to pH 8.0 with saturated aquueous lithium carbonate or 1N potassium hydroxide.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Helly and Bouin are also suitable. Other fixatives should be tested.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into freshly pH adjusted staining solution for 30-60 minutes.
  3. Wash with running tap water for 10 minutes.
  4. Dehydrate with ethanols.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  blue
  • Collagen  –  pale violet

Notes

  • Note that this is an ethanolic variant of Mayer’s hemalum.
  • If sodium iodate is left out, the staining solution lasts 3-4 days, but the staining time is extended to 60 minutes.
  • The solution may also be made by diluting 30 mL Mayer’s hemalum with 70 mL absolute ethanol, mixing for 20 minutes, and filtering before adjusting the pH. Staining requires 60 minutes and the results are paler.

Safety Note

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

References

  1. Akita, M. and Kaneko, K., (1981)
    An improved hematoxylin method with application of Mayer’s hemalum for staining of elastic fibres.
    Stain Technology, v. 56, p. 327
  2. Kaneko, K. and Akita, M., (1978)
    On the staining of elastic fibres with alum hematoxylin.
    Stain Technology, v. 53, p. 43.
March 30, 2023
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McNulty-Smith’s Zirconyl Hematoxylin

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

McNulty-Smith's Zirconyl Hematoxylin

6
steps
6
materials
print

This hematoxylin substitutes for alcian blue pH 2.5 for staining acid mucins.

Materials

IngredientAmountFunction
Hematoxylin100 mgDye
Ethanol, absolute5 mLSolvent
Sodium iodate, 0.5% aqu.5 mLOxidant
Zirconyl chloride, octahydrate400 mgMordant
Distilled Water22.5 mLSolvent
Glycerol7.5 mLAnti-oxidant

Compounding Procedure

  1. Dissolve the hematoxylin in the ethanol.
  2. Add the sodium iodate solution (freshly made).
  3. Add the zirconyl chloride.
  4. Combine the glycerol and water, and add to the solution.
  5. Stir for 5 minutes.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 10 minutes.
  3. Wash with distilled water, two changes of 2 minutes each.
  4. Counterstain in methylene green for 5 minutes.
  5. Wash with distilled water, two changes of 2 minutes each.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  green
  • Acid mucins  –  Purple

Notes

  • Formalin fixed, paraffin sections are suitable.
  • The counterstain recommended was 0.05% methylene green in 2.4% boric acid.
  • This method was recommended as a substitute for alcian blue staining of acid mucins (pH 2.5).
  • Staining may be removed with 1% hydrochloric acid in 70% ethanol.
  • The solution above is McNulty’s modification of Smith’s original, and stains more intensely:
    • Hematoxylin, 100 mg
    • Absolute ethanol, 5 mL
    • 0.1% aqueous sodium iodate, 5 mL
    • Zirconyl chloride octahydrate, 400 mg
    • 22% aqueous glycerol, 45 mL
    • Glacial acetic acid, 5 mL

Safety Note

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

References

  1. Smith, A. A., (1999)
    Zirconyl hematoxylin staining of acidic mucins.
    Journal of Histochemistry and Cytochemistry, v. 47, p. 1645.
  2. McNulty, J. M. and Smith, A. A., (2004)
    An improved formulation of the zirconyl hematoxylin stain for acidic mucins.
    Biotechnic and Histochemistry, v. 79, Nº 5, p. 191.
  3. McNulty, J. M., Kambour, M. J. and Smith, A. A., (2004)
    Use of an improved zirconyl hematoxylin stain in the diagnosis of Barrett’s esophagus.
    J. Cell. Mol. Med., v. 8, Nº 3, p. 382.
March 30, 2023
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Mayer’s Alum Hematoxylin Staining Variants

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

The solution usually meant when Mayer’s hemalum is specified is actually a modification of Mayer’s 1901 formula by Langeron. Mayer published several alum hematoxylin variants for nuclear staining, both progressive and regressive. Such solutions usually contain hematoxylin and an alum, and are called hemalum or alum hematoxylin solutions. Many formulae have been suggested. They vary in the amount of hematoxylin, the amount and type of aluminum salts, solvents, oxidizing agents and stabilizers.

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March 30, 2023
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