Category

Dye Type

Graham’s Aluminum Hematoxylin

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

Graham's Aluminum Hematoxylin

6
steps
5
materials
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Grahams Alum hematoxylin is a modification of Gill’s Alum hematoxylin, and was designed for routine staining of plant tissue. It should also be satisfactory for animal tissues. It has been formulated so that small quantities may be prepared fresh and sufficient for a single Coplin jar.

Materials

MaterialAmountFunction
Distilled water750 mLSolvent
Propylene glycol210 mLSolvent
Glacial acetic acid20 mLAcidifier
Aluminum sulphate17.6 gMordant
Sodium iodate0.2 gOxidant
MaterialAmountFunction
Solution BAmountFunction
Hematoxylin10 gDye
Propylene glycol100 mLSolvent

Compounding Procedures

Solution A

  1. Combine all the ingredients and filter when dissolved.

Solution B

  1. Shake vigorously several times during 48 hours until the hematoxylin dissolves.

Working solution

  1. Solution A – 49 mL
  2. Solution B – 1 mL
  3. Mix well.
  4. It is ready for use when an opaque dark purple.

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

  • Staining is progressive, application time being determined by trial (try 2 minutes).
  • Propylene glycol replaces toxic ethylene glycol.
  • 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.
  • The aluminum sulphate should be Al2(SO4)3.18H2O. Adjust the amount if different.

Safety Note

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

References

  1. Graham, E. T., (1991)
    A quick mix aluminum hematoxylin stain
    Biotechnic and Histochemistry, pp. 279-281
March 30, 2023
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Hamilton Alum Hematoxylin

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

Hamilton Alum Hematoxylin

6
steps
6
materials
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This formula is based on Langeron’s 1942 (Mayer’s) formula. It has been referred to as Hamilton Alum hematoxylin as it was received via the internet from a histotechnologist at the Hamilton Health Sciences Corporation, Hamilton, Ontario, Canada.

Materials

MaterialAmountFunction
Hematoxylin2 gDye
Ammonium alum75 gMordant
Distilled water950 mLSolvent
100% ethanol50 mLSolvent
Sodium iodate0.3 gOxidant
Glacial acetic acid30 mLAcidifier

Compounding Procedure

  1. Dissolve the Alum in the water.
  2. Add the ethanol and mix well.
  3. Add the hematoxylin and dissolve.
  4. Add the sodium iodate and mix well.
  5. Leave overnight in the dark to ripen.
  6. Mix well, and add the acetic acid.
  7. Filter.
  8. The solution may be used immediately.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 3 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

  • Potassium Alum may be used instead of ammonium alum.
  • The solution is progressive.
  • 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

Personal internet communication, source unidentified.

March 30, 2023
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Hansen’s Iron Hematoxylin

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

Hansen's Iron Hematoxylin

8
steps
3
materials
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Materials

MaterialAmountFunction
Hematoxylin0.75 gDye
Ferric ammonium sulfate4.5 gMordant
Distilled water100 mLSolvent

Compounding Procedure

  1. Dissolve the iron alum in 65 mL of the water.
  2. Dissolve the hematoxylin in the remaining water.
  3. Combine the solutions and bring to a boil.
  4. Cool and filter.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse with tap water.
  4. Treat 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 stability of the solution is not given, but the simplicity of the formula indicates it would not be stable for too long.
  • Although a staining time was not given, 30 minutes should be sufficient. Determine 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:
    Hansen, (1905)
    Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik,
    v.22, p.55. Leipzig.
March 30, 2023
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Harris’ Alum Hematoxylin

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

Harris' Alum Hematoxylin

8
steps
6
materials
print

Materials

MaterialAmountFunction
Hematoxylin5 gDye
Potassium alum100 gMordant
Distilled water1 LSolvent
95% ethanol50 mLSolvent
Mercuric oxide2.5 gOxidant
Glacial acetic acid40 mLAcidifier

Compounding Procedure

  1. Dissolve the hematoxylin in the ethanol.
  2. Dissolve the Alum in warmed distilled water.
  3. Combine the solutions in an oversize container.
  4. Bring to a boil.
  5. Add the mercuric oxide, remove from heat.
  6. Cool rapidly.
  7. Add the glacial acetic acid.
  8. It may be used immediately when cool.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Paraffin sections should be stained for 5 minutes, and cytology smears for 45 seconds.
  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 solution is a regressive formulation, although it is sometimes used progressively with short staining times.
  • When the mercuric oxide is added, the solution may foam.
  • Mercuric oxide is toxic and no longer recommended for oxidation of hematoxylin. If used, all discarded solution should be disposed of as mercury contaminated. Sodium iodate is sometimes substituted, based on a maximum of 0.2 gram sodium iodate for each 1 gram of hematoxylin. In practice, 0.5 gram works quite well.
  • The acetic acid is optional, but nuclear staining is more precise if it is included.
  • A precipitate often forms, so the solution should be filtered before use.
  • As with many strong hematoxylin solutions, mucin 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

  1. Culling, C.F.A., Allison, R.T. and Barr, W.T.
    Cellular Pathology Technique, Ed.4.
    Butterworth, London, UK.
  2. Drury, R.A.B. and Wallington, E.A., (1980)
    Carleton’s histological technique Ed. 5
    Oxford University Press, Oxford, UK.
  3. 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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Harris & Power’s Alum Hematoxylin

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

Harris & Power's Alum Hematoxylin

8
steps
4
materials
print

Materials

MaterialAmountFunction
Hematoxylin20 gDye
Potassium alum60 gMordant
Distilled water100 mLSolvent
100% ethanol6 mLSolvent

Compounding Procedure

  1. Mix the Alum and hematoxylin in a mortar.
  2. Add the water, little by little, while grinding the mixture.
  3. Filter, and add the ethanol.

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

  • This very strong mixture should, presumably, be left to ripen for some time.
  • Its characteristics and recommended use were not given.
  • 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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Haug’s Alum Hematoxylin

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

Haug's Alum Hematoxylin

8
steps
4
materials
print

Materials

MaterialAmountFunction
Hematoxylin5.5 gDye
Aluminum acetate5 gMordant
Distilled water100 mLSolvent
100% ethanol5 mLSolvent

Compounding Procedure

  1. Dissolve the hematoxylin in ethanol.
  2. Dissolve the aluminum acetate in water.
  3. Combine.

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

  • This strong formula should, presumably, be left for some time to ripen.
  • The characteristics and recommended use were not given.
  • 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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Heidenhain’s Iron Hematoxylin

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

Heidenhain's Iron Hematoxylin

8
steps
4
materials
print

Materials

Solution A

MaterialAmountFunction
Ferric ammonium sulfate2.5 gMordant
Distilled water100 mLSolvent

Solution

MaterialAmountFunction
Hematoxylin0.5 gDye
Distilled water100 mLSolvent

Compounding Procedure

  1. Make each solution separately.
  2. Solution B should be ripened for a minimum of one month.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into solution A for 30 minutes to 24 hours.
  3. Rinse with distilled water.
  4. Place into solution B for 30 minutes to 24 hours.
  5. Rinse with tap water.
  6. Differentiate in solution A, controlling microscopically.
  7. Wash well in running tap water to blue.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei and other structures  –  blue to black

Notes

  • The stock solutions are stable for some time.
  • The hematoxylin solution needs to be ripened.
  • Staining at elevated temperatures (not over 60°C) will shorten the required times. Always differentiate at room temperature.
  • The degree of differentiation will determine which tissue components are prominent. The method can demonstrate many structures, including chromosomes, nuclear components, mitochondria and muscle striations
  • The solutions may be reused, with the exception of solution A, which is used to differentiate, which should be fresh each time.
  • Counterstaining is not recommended.
  • This method is usually recommended for monochrome photography.
  • Compare Heidenhain’s iron hematoxylin technique to other published variations.

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:
    Heidenhain, M., (1892).
    Festschrift Herrn A. von Kolloker zur Feier seines
    fünfzigjährigen medicinischen
    Doktorjubiläums, p.118.
    Wilhelm Engellmans, Leipzig, Germany
March 30, 2023
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Heidenhain’s Vanadium Hematoxylin

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

Heidenhain’s Vanadium Hematoxylin

4
steps
3
materials
print

Materials

Stock A

MaterialAmountFunction
Hematoxylin0.5 gDye
Distilled water100 mLSolvent

Stock B

MaterialAmountFunction
Ammonium vanadate0.25 gMordant
Distilled water100 mLSolvent

Working Solution

MaterialAmountFunction
Stock A60 mLDye
Stock B30 mLMordant

Compounding Procedure

  1. Make each stock solution separately.
  2. Combine in the specified proportions.

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. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • No details were given.

Notes

  • 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:
    Böhm and Oppel, (1907)
    Manuel de technique microsopique, ed. 4, p. 105.
    Vigot, Paris, France
March 30, 2023
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Held’s Molybdenum Hematoxylin

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

Held's Molybdenum Hematoxylin

6
steps
6
materials
print

Materials

Stock Solution

MaterialVariation IVariation IIFunction
Hematoxylin1 g1 gDye
Phosphomolybdic acid15 g–Mordant
Molybdic acid–excessMordant
Distilled water30 mL30 mLSolvent
95% ethanol70 mL70 mLSolvent

Solution A

MaterialAmountFunction
Ferric ammonium sulfate5 gMordant
Distilled water100 mLSolvent

Solution B

MaterialAmountFunction
Stock solution5 mL–
Distilled water95 mLSolvent

Compounding Procedures

Stock solution

  1. Mix the water and ethanol together.
  2. Dissolve the hematoxylin.
  3. Add the molybdenum compound specified and shake frequently over a period of one month until there is a distinct colour change to a deep blue-black, then decant.
  4. The staining improves with aging.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Optionally, place into solution A for 24 hours.
  3. Place into solution B for 12-24 hours.
  4. Differentiate with solution A if overstained.
  5. Wash in running tap water.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Tissue components  –  various shades.

Notes

  • Variation I is from the Microtomist’s Formulary and Guide and specifies phosphomolybdic acid, variation II is from the Microtomists Vade Mecum and specifies molybdic acid.
  • It is recommended for developing nerve tissues.

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:
    Bolles-Lee, A.; Edited by Gatenby, J. B. & Painter, T. S., (1937)
    The microtomists’s Vade-Mecum, p. 374
    Blakiston, Philadelphia, USA
  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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Shum & Hon’s PTAH

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

Shum & Hon's PTAH

6
steps
3
materials
print

Materials

MaterialAmountFunction
Hematein,0.8 g.Dye
Phosphotungstic acid9 gMordant
Distilled water1 LSolvent

Compounding procedures

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

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

  • 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

  1. Histological demonstration techniques, (1974)
    Cook, H C.
    Butterworths, London, England.
March 30, 2023
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