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Hematoxylin and Eosin Staining

Lillie & Earle’s Iron Hematoxylin

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

Lillie & Earle's Iron Hematoxylin

8
steps
6
materials
print

Materials

Solution A

MaterialAmountFunction
Hematoxylin1 gDye
95% ethanol50 mLSolvent
Glycerol50 mLSolvent

Solution B

MaterialAmountFunction
Ferric ammonium sulfate15 gMordant
Ferrous sulfate15 gMordant
Distilled water100 mLSolvent

Compounding Procedure

  1. Make each solution separately.
  2. For use, combine equal parts of solutions A and B.
  3. The working solution 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 with tap water.
  4. If overstained, 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 life of the working solution is not given. It is likely not stable for long, although the inclusion of glycerol and ferrous sulfate may improve its stability.
  • The staining time should be determined by trial, but 10-30 minutes should suffice.

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:
    Lillie and Earle, (1937).
    American Journal of Pathology, v. 15, p. 765.
March 30, 2023
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La Manna’s Iron Hematoxylin

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

La Manna's Iron Hematoxylin

8
steps
3
materials
print

Materials

MaterialAmountFunction
Hematoxylin1 gDye
Ferric ammonium sulfate3 gMordant
Distilled water100 mLSolvent

Compounding Procedure

  1. Dissolve the iron alum in half the water.
  2. Dissolve the hematoxylin in the rest.
  3. Combine.

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. 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 simplicity of the formula suggests the solution is not stable for long.
  • 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:
    La Manna, (1937)
    Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik,
    v. 22, p. 55. Leipzig.
March 30, 2023
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Langeron’s Alum Hematoxylin Variants

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

Langeron's Alum Hematoxylin Variants

8
steps
9
materials
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Both of Langeron’s formulae are modifications of Mayer’s formulae. Langeron’s 1924 formula is a modification of Mayer’s 1896 formula, and Langeron’s 1942 formula is a modification of Mayer’s 1901 formula.

Langeron’s 1942 formula is the solution usually meant when Mayer’s hemalum is specified.

Materials

MaterialSolutionFunction
19241942
Hematoxylin4 g1 gDye
Ammonium alum50 g–Mordant
Potassium alum–50 gMordant
Distilled water700 mL1 LSolvent
Glycerol300 mL–Stabilizer
Sodium iodate–0.2 gOxidant
Glacial acetic acid20 mL–Acidifier
Citric acid–1 gAcidifier
Chloral hydrate–50 gAcidifier

Compounding Procedures

1924

  1. Grind the hematoxylin with glycerol in a pestle and mortar.
  2. Dissolve the other ingredients in water.
  3. Add the hematoxylin paste.
  4. Wash out the hematoxylin paste with the solution.
  5. Leave months to ripen.

1942

  1. Dissolve the Alum and dye in the water.
  2. Add the other ingredients.
  3. Bring to the boil.
  4. Cool to room temperature.
  5. The solution may be used immediately.
  6. Ammonium Alum may be substituted for potassium alum.

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 the 1924 solution 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 1924 solution is regressive, and staining time should be determined by trial.
  • The 1942 solution is progressive. Staining time is 5-10 minutes.
  • 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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Launoy’s Alum Hematoxylin

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

Launoy's Alum Hematoxylin

8
steps
3
materials
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Materials

MaterialAmountFunction
Hematein10 gDye
Potassium alum5 gMordant
Distilled water1 LSolvent

Compounding Procedure

  1. Combine all ingredients, and dissolve.
  2. The solution 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

  • This solution uses hematein instead of hematoxylin.
  • Although the staining characteristics are not given, the high dye and low Alum content would indicate it is likely a strong, regressive solution.
  • 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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Lee’s Alum Hematoxylin

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

Lee's Alum Hematoxylin

6
steps
5
materials
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Materials

MaterialAmountFunction
Hematoxylin1 gDye
Ammonium alum50 gMordant
Distilled water1 LSolvent
Chloral hydrate50 gStabiliser
Sodium iodate0.2 gOxidant

Compounding Procedure

  1. Combine all the ingredients.
  2. Bring to a boil.
  3. Cool and filter.
  4. The solution 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 solution is similar to Mayer’s hemalum and should have much of the same characteristics.
  • 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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Lillie’s Alum Hematoxylin

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

Lillie's Alum Hematoxylin

8
steps
6
materials
print

Materials

MaterialAmountFunction
Hematoxylin5 gDye
Ammonium alum50 gMordant
Distilled water700 mLSolvent
Glycerol300 mLStabilizer
Sodium iodate0.5 gOxidant
Glacial acetic acid20 mLAcidifier

Compounding Procedure

  1. Dissolve the dye and Alum in the water.
  2. Add the iodate, glycerol and acid.
  3. Leave overnight to ripen before using.

Protocol

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

  • This is a strong, regressive solution.
  • 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. Lillie, R.D.,
    Histopathologic Technic and Practical Histochemistry
    McGraw-Hill, New York, USA
March 30, 2023
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Lillie’s Iron Hematoxylin

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

Lillie's Iron Hematoxylin

6
steps
6
materials
print

Materials

MaterialVariantFunction
19401954
Hematoxylin1 g0.5 gDye
Ferric chloride1.2 g1.2 gMordant
Ferrous sulfate–4.44 gMordant
100% ethanol–100 mLSolvent
Distilled water100 mL292 mLSolvent
Hydrochloric acid1 mL8 mLSolvent

Compounding Procedures

1940

    1. Dissolve the ferric chloride in half of the water.
    2. Dissolve the hematoxylin in the other half.

Combine and add the hydrochloric acid.

1954

  1. Dissolve the hematoxylin in the ethanol.
  2. Dissolve the ferric chloride and ferrous sulfate in the water.
  3. Add the hydrochloric acid.
  4. Combine the solutions.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 5-30 minutes.
  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 1940 formula is likely not stable for long. If staining is too dark, dip briefly in 1% hydrochloric acid in 70% ethanol.
  • The 1954 formula is a modification of Wiegert’s iron hematoxylin. Lillie states that it is stable for several weeks with occasional use. It is progressive, and does not require differentiation.

Safety Note

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

References

  1. Lillie, R.D., (1954)
    Histopathologic technique and practical histochemistry Ed.2
    Blakiston, New York, USA.
  2. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    Lillie, (1940)
    Archiv für pathologische Anatomie,
    v. 29, p. 705.
March 30, 2023
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Lugol’s Alum Hematoxylin

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

Lugol’s Alum Hematoxylin

6
steps
6
materials
print

Materials

Lugol’s Iodide

MaterialAmountFunction
Iodine2 gOxidant
Potassium iodide4 g
Distilled water100 mLSolvent

Hematoxylin

MaterialAmountFunction
95% ethanol100 mLSolvent
Hematoxylin10 gDye

Alum

MaterialAmountFunction
Potassium alum6 gMordant
Distilled water500 mLSolvent

Working solution

MaterialAmount
Hematoxylin1.5 mL
Lugol’s iodine1.5 mL
Alum100 mL

Compounding Procedure

  1. Make each solution as listed.
  2. Combine as directed above.
  3. Leave for 15 minutes before use.

Protocol

  1. Bring sections to distilled 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

  • Read these comments on Lugol’s iodine.
  • The solution is usable for a minimum of one week.
  • The working solution will stain at least 100 slides.
  • Since the solution is not acidified, a distilled water rinse before staining will reduce alkaline carry over.
  • The authors named this solution Lugol’s Hematoxylin.
  • 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. Clarke, G. and Dodds, H. M., (1983)
    Lugol’s Hematoxylin.
    Stain Technology, v 58, Nº 4, p. 232
March 30, 2023
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McLachlan’s Alum Hematoxylin

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

McLachlan's Alum Hematoxylin

6
steps
7
materials
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This formulation has been named in honor of Dr. H.K.I. McLachlan FRCPA at the request of the originator, Mike Rentsch.

Materials

MaterialVariantFunction
Var IVar II
Hematoxylin2 g2 gDye
Aluminum sulphate17.5 g–Mordant
Ammonium alum–25 gMordant
Distilled water700 mL700 mLSolvent
Glycerol300 mL300 mLStabiliser
Sodium iodate0.2 g0.2 gOxidant
Glacial acetic acid20 mL20 mLAcidifier

Compounding Procedure

  1. Dissolve the aluminum sulphate or the Alum in about 500 mL water.
  2. Add the hematoxylin and dissolve.
  3. Add the acetic acid and mix well.
  4. Warm the glycerol to reduce viscosity, and add to the mixture.
  5. Mix well, then make up to 1 litre with water.
  6. Add the iodate and mix for 5-10 minutes.
  7. Store in a tightly capped bottle in the dark.
  8. The solution remains usable for 2-5 years.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place paraffin sections in the staining solution for 2-4 minutes and frozen sections for 40-60 seconds.
  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

  • This is a 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. Rentsch, M.
    Australian Biostain P/L.
    Personal communication via the internet.
March 30, 2023
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Janssen’s Iron Hematoxylin

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

Janssen's Iron Hematoxylin

8
steps
6
materials
print

Materials

MaterialAmountFunction
Hematoxylin1 gDye
Ferric ammonium sulfate5 gMordant
100% ethanol5 mLSolvent
Distilled water70 mLSolvent
Glycerol15 mLSolvent
Methanol15 mLSolvent

Compounding Procedure

Gray Method

  1. Dissolve the iron alum into the water.
  2. Dissolve the hematoxylin into the ethanol.
  3. Combine, and leave for one week at room temperature.
  4. Filter, and then add the other ingredients.

Lillie Method

  1. Combines all solutions immediately once they are dissolved, and omits the week at room temperature.
  2. The solution is stable for a few months.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 15-30 minutes.
  3. Rinse with tap water.
  4. 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

  • This solution may be used as an acid resistant nuclear stain.
  • It is recommended as a substitute for Weigert’s iron hematoxylin.

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:
    Janssen, (1897),
    Cellule, v.14, p.207.
    Lillie & Earle, (1939),
    Stain Technology, v.14, p.53
March 30, 2023
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