Category

Dye Type

Friedlander’s Alum Hematoxylin

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

Friedlander's Alum Hematoxylin

8
steps
5
materials
print

Materials

MaterialAmountFunction
Hematoxylin6 gDye
Potassium alum6 gMordant
Distilled water300 mLSolvent
95% ethanol300 mLSolvent
Glycerol300 mLStabiliser

Compounding procedures

  1. Dissolve the Alum in the water.
  2. Dissolve the hematoxylin in the ethanol.
  3. Combine, and 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

  • This is probably a regressive formula.
  • 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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Gadsdon’s Alum Hematoxylin

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

Gadsdon's Alum Hematoxylin

8
steps
7
materials
print

This formula is by Prof. Derek Gadsdon at the Sheffield Children’s Hospital, UK.

Materials

MaterialAmountFunction
Hematoxylin5.5 gDye
Potassium alum60 gMordant
Distilled water610 mLSolvent
100% ethanol100 mLSolvent
Glycerol300 mLStabiliser
Sodium iodate0.5 gOxidant
Glacial acetic acid20 mLAcidifier

Compounding procedures

  1. Dissolve the hematoxylin in 100 mL ethanol.
  2. Dissolve the Alum in 600 mL water.
  3. Dissolve the sodium iodate in 10 mL water.
  4. Combine the hematoxylin and Alum solutions. Mix well.
  5. Add the sodium iodate solution. Mix well.
  6. Add the acetic acid. Mix well.
  7. Leave overnight.
  8. Add the glycerol. Mix well.
  9. Filter before use.
  10. The solution may be used immediately.
  11. It is stable at room temperature for at least 6 months.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for the appropriate time.
  3. Rinse well with water.
  4. Differentiate with acid ethanol if using regressively.
  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 may be used regressively or progressively.
    • Regressively, staining time is 6 minutes.
    • Progressively, staining time is 3 minutes.
    • Frozen sections will stain in 1 minute.
  • As with many strong formulations mucins may stain 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. Histopathology laboratory, Red Cross Children’s Hospital.
    Gadsdon’s Haematoxylin Method
    Histonet communication, Nov, 1996
  2. Jim Elsam, HTEQA Services.
    Gadsdon’s Hx
    Histonet communication, Nov, 1996
March 30, 2023
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Gage’s Alum Hematoxylin

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

Gage's Alum Hematoxylin

6
steps
5
materials
print

Materials

MaterialAmountFunction
Hematoxylin1 gDye
Potassium alum40 gMordant
Distilled water1 LSolvent
95% ethanol20 mLSolvent
Chloral hydrate20 gOxidant

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 formula indicates the solution is progressive.
  • As no oxidising agent is present the solution needs ageing.
  • The appropriate time should be determined by trial, but 5-10 minutes should be adequate.
  • 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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Galigher’s Alum Hematoxylin

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

Galigher's Alum Hematoxylin

8
steps
5
materials
print

This solution was recommended as a substitute for Harris’ Alum hematoxylin.

Materials

MaterialAmountFunction
Hematoxylin5 gDye
Ammonium alum3 gMordant
Distilled water500 mLSolvent
100% ethanol500 mLSolvent
Mercuric oxide6 gOxidant

Compounding procedures

  1. Combine the water and ethanol.
  2. Add the Alum and dye, and bring to a boil.
  3. Add the mercuric oxide, then simmer for 20 minutes.
  4. Restore the volume to 1 L with 50% ethanol.
  5. Cool, and filter through double thickness filter paper.
  6. Store in a tightly stopper bottle.
  7. The solution may be used immediately, and is stable for about six months.

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

  • Mercuric salts are toxic and no longer recommended for oxidation of hematoxylin. Ensure that discarded solution is disposed of as mercury contaminated, or substitute 0.5 grams sodium iodate as the oxidant.
  • Staining capability may continue to improve for a short while.
  • 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. 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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Garvey’s Alum Hematoxylin

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

Garvey's Alum Hematoxylin

6
steps
6
materials
print

Materials

MaterialAmountFunction
Hematoxylin2.5 gDye
Potassium alum45 gMordant
Distilled water900 mLSolvent
100% ethanol100 mLSolvent
Sodium iodate0.3 gOxidant
Citric acid1 gAcidifier

Compounding procedures

  1. Dissolve the hematoxylin in the ethanol.
  2. Dissolve the Alum in the distilled water with heat.
  3. Combine the two solutions, then add the sodium iodate and citric acid.
  4. Shake well to dissolve.
  5. The solution may be used immediately, and is stable for several months.

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

  • Ammonium Alum may be used instead of potassium alum.
  • As the solution was recommended as a substitute for Mayer’s Alum hematoxylin, staining times and results should be comparable. However, the increased hematoxylin content indicates the solution should stain more darkly.
  • 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. Winsome Garvey,
    Modification of the Mayer Hematoxylin stain,
    Journal of Histotechnology, v.14, No.3, p.163
March 30, 2023
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Gallego’s Carbol Fuchsin for Nuclei

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

Gallego's Carbol Fuchsin

for Nuclei

8
steps
3
materials
print

Materials

Solution A

MaterialAmount
Carbol fuchsin2mL
Distilled water98mL

Solution B

MaterialAmount
Formalin, conc. (40%)1mL
Distilled water99mL

Tissue Sample

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

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into solution A for 5 minutes.
  3. Wash with water.
  4. Place into solution B for 5 minutes.
  5. Wash with water.
  6. Stain with a contrast stain.
  7. Dehydrate with ethanol.
  8. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue-black
  • Cytoplasm  –  as counterstain

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:
    Gallego, (1919)
    Trabajos del Laboratorio de investigaciones biológicas de la
    Universidad de Madrid,
    v. 17, pp.95
    And:
    Biot, (1910)
    Compte rendu de l’Association des anatomistes, Congrès de Lyon, pp.234
March 30, 2023
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Duval’s Alum Hematoxylin

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

Duval's Alum Hematoxylin

6
steps
4
materials
print

Materials

Original Formula

MaterialAmountFunction
Hematoxylin, concentrated ethanolic8 mLDye
Ammonium or potassium aluma littleMordant
Distilled water800 mLSolvent

Modern Formula

MaterialAmountFunction
Hematoxylin, saturated ethanolic8 mLDye
Ammonium or potassium alum25 gMordant
Distilled water800 mLSolvent

Compounding procedure

  1. Dissolve the alum in the water.
  2. Add the hematoxylin.
  3. The solution is likely progressive, although this is not stated to be so.

Protocol

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

  • This solution is from the late 1800’s and is now obsolete, although the modern formula should stain satisfactorily.
  • Concentrated alcoholic hematoxylin, after ripening, would have contained no more than 7% hematein. It was made by soaking logwood chips in ethanol until no more dye dissolved out, then oxidized naturally. Depending on the sample of logwood and the amount of dye it contained, more than one batch may have been necessary to saturate the ethanol.
  • The type of alum was not specified, the most likely being either potassium or ammonium.
  • The formula calls for adding a “little alum” to 800 mL water. Potassium alum saturates at about 14% in water, so 800 mL would contain about 112 g. I have taken just less than 25% of the maximum (i.e. 25 grams) as being a “little”.
    Of course, it could be any amount between 1 and 112 grams.
  • The appropriate time should be determined by trial. The instructions are to use full strength for a few minutes.
  • 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. Arthur Bolles-Lee, (1885)
    The Microtomist’s Vade-Mecum
    Originally published by: J & A Churchill, London, England.
    Republished by: Science Heritage Ltd., Lincolnwood, Illinois, USA.
  2. Susan Budavari, Editor, (1996)
    The Merck Index, Ed. 12
    Merck & Co., Inc., Whitehouse Station, NJ, USA
March 30, 2023
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Ehrlich’s Alum Hematoxylin

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

Ehrlich's Alum Hematoxylin

8
steps
6
materials
print

Materials

MaterialAmountFunction
Hematoxylin6 gDye
95% ethanol300 mLSolvent
Potassium alumexcessMordant
Distilled water300 mLSolvent
Glycerol300 mLStabiliser
Glacial acetic acid30 mLAcidifier

Compounding procedures

  1. Dissolve the hematoxylin in the ethanol mixed with acetic acid.
  2. Dissolve the alum in the water mixed with glycerol in an oversized container.
  3. Add the hematoxylin solution to the alum solution.
  4. Plug the container loosely with cotton wool.
  5. Ripen by leaving in a warm, sunlit place for several weeks.
  6. When sufficiently ripened, store tightly stoppered in a cool, dark place.
  7. The solution is stable for years.

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

  • Gray gives 7 grams hematoxylin, and specifies ammonium alum.
  • The alum should be added to excess. This should be about 50 grams, but enough should be added to ensure undissolved alum is present.
  • This is a strongly staining, regressive formula. The staining time should be determined by trial. Usually, 20 minutes is adequate.
  • As with many strong alum hematoxylin solutions, cartilage, cement lines and mucin may stain blue.
  • The solution may be chemically ripened by adding 0.5g sodium iodate, but chemicallly ripened solutions are inferior in longevity.
  • 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.
  4. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    Ehrlich, (1886)
    Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik, v. 3, p. 150.
    Leipzig.
March 30, 2023
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Verhoeff’s Iron Hematoxylin for Elastic

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

Verhoeff's Iron Hematoxylin

for Elastic

10
steps
7
materials
print

Materials

Stock Verhoeff A

MaterialAmount
Hematoxylin1g
Ethanol, absolute20mL

Stock Verhoeff B

MaterialAmount
Ferric chloride10g
Distilled water100mL

Stock Verhoeff C

MaterialAmount
Potassium iodide4g
Iodine2g
Distilled water100mL

Working solution

MaterialAmount
Stock solution A20mL
Stock solution B8mL
Stock solution C8mL

Differentiator

MaterialAmount
Stock solution B10mL
Distilled water40mL

Tissue Sample

A 10% Formalin variant is suitable. Paraffin sections at 5µ are preferred.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place in working Verhoeff’s solution for 15 minutes.
  3. Wash well with tap water to remove all excess hematoxylin.
  4. Differentiate until the elastic fibres are satisfactory. This should be controlled microscopically based on the appearance of fibres in the area of interest.
  5. Rinse well with tap water.
  6. Place into 95% ethanol for 5 minutes to remove iodine discolouration.
  7. Wash well with tap water to remove all residual chemicals.
  8. Counterstain with Van Gieson.
  9. Dehydrate with absolute ethanol.
  10. Clear with xylene and mount with a resinous medium

Expected Results

  • Nuclei  –  black
  • Elastic fibres  –  black
  • Muscle  –  yellow
  • Collagen  –  red

Notes

  • The alcoholic hematoxylin solution should be freshly made. It does not need to have been ripened as ferric chloride is an oxidising agent. Some technologists keep pre-weighed aliquots of 1 gram hematoxylin and dissolve it in 20 mL ethanol as needed.
  • The working solution should be made just before use and allowed to stand for five minutes to ripen.
  • While this is a popular elastic stain, it is difficult to stain both coarse fibres and fine fibres optimally in a single section. If coarse fibres are well stained, the finest fibres may be completely decolourised, and if fine fibres are optimally stained, the coarse fibres are underdifferentiated. Ensure differentiation is optimised for the fibres of interest.
  • If Van Gieson is used as a counterstain, the elastic fibres should be very slightly underdifferentiated as picric acid will also remove some hematoxylin staining.
  • This method likely stains by two mechanisms: ionic attachment of iron mordanted hematoxylin to most structures and by van der Waal’s forces to elastic fibres. The differentiation step is accomplished either by oxidative bleaching of hematoxylin by ferric chloride, or by displacement of the mordanted hematoxylin by free mordant in solution. The dye attached to elastic fibres by van der Waal’s forces is not extracted easily. It is, however, still susceptible to bleaching, which likely explains why fine fibres may be pale. That bleaching takes place is shown by the differentiating fluid remaining clear during differentiation, extracted dye being seen to bleach as it dissolves out.
  • Note that nuclei are only adequately, not well, stained.

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
  2. Culling, C.F.A., (1963)
    Handbook of histopathological techniques, 2nd ed.
    Butterworths, London.
March 30, 2023
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Taenzer-Unna Orcein for Elastic Fibres

By Dye Type, Elastic Fibers, Orcein, Protocols, Stain Target, Stain Type
Protocol

Taenzer-Unna Orcein

for Elastic Fibres

8
steps
3
materials
print

Materials

Orcein solution

MaterialAmount
Orcein1g
Ethanol, 70%100mL
Hydrochloric acid, conc.1mL

Acid ethanol

MaterialAmount
Ethanol, 70%100mL
Hydrochloric acid, conc.1mL

Tissue Sample

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

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Rinse with 70% ethanol.
  3. Place into orcein solution for an appropriate time.
  4. Optionally, treat with acid ethanol until collagen is unstained (a few seconds).
  5. Wash with water
  6. Counterstain if wished.
  7. Dehydrate with 95% and absolute ethanols.
  8. Clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  purplish brown
  • Other tisse  –  as counterstained

Notes

  • Gray specifies 0.8 grams orcein, most others specify 1 gram.
  • Gray specifies 50% ethanol, Culling 70% and Carleton 80%. Others have recommended concentrations ranging from 65% to 100% (Lillie).
  • Staining times specified include 6-12 hours (Gray), overnight (Culling) and 30 minutes to 2 hours (Carleton).
  • Elevating the temperature (37°C Carleton, 56°C Culling) decreases staining time.
  • Recommended counterstains are van Gieson, methylene blue, H&E.

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:
    von Kahlden, C., and Laurent, O. (1896)
    Technique microscopique.
    Carré, Paris, France
  2. Drury, R.A.B. and Wallington, E.A., (1980)
    Carleton’s histological technique Ed. 5
    Oxford University Press, Oxford, UK.
  3. Culling C.F.A., (1974)
    Handbook of histopathological and histochemical techniques Ed. 3
    Butterworth, London, UK.
  4. Mallory, F. B. & Wright, J.H., (1904)
    Pathological technique, Ed.3
    W. B. Saunders, Philadelphia, USA.
  5. Lillie, R.D., (1954)
    Histopathologic technique and practical histochemistry Ed.2
    Blakiston, New York, USA.
March 30, 2023
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