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Hematoxylin and Eosin Staining

Celestine Blue Hemalum An Acid Resistant Nuclear Stain

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

Celestine Blue Hemalum

An Acid Resistant Nuclear Stain

6
steps
2
materials
print

Materials

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. Place into the iron alum-celestine blue solution for 10 minutes.
  3. Wash with tap water.
  4. Place into the alum hematoxylin for 10 minutes.
  5. Wash with water to blue.
  6. Continue with the counterstain

Expected Results

  • Nuclei – blue-black
  • Other tissue – according to the counterstain

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. Bancroft, J.D. and Stevens A. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
  3. Humason, G. L., (1967)
    Animal tissue techniques, Ed. 2
    W. H. Freeman and Company, San Francisco, Ca, USA
  4. Drury, R.A.B. and Wallington, E.A., (1980)
    Carleton’s histological technique Ed. 5
    Oxford University Press, Oxford, UK.
  5. Putt, F. A.
    Manual of Histopathological Staining Methods
    John Wiley & Sons, New York, NY., USA
March 30, 2023
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Crétin’s Iron Hematoxylin

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

Crétin's Iron Hematoxylin

8
steps
6
materials
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Materials

Solution A

MaterialAmountFunction
Ferrous sulfate4 gMordant
Distilled water100 mLSolvent

Solution B

MaterialAmountFunction
Potassium ferrocyanide2 gMordant
Potassium ferricyanide1 gMordant
Distilled water100 mLSolvent

Solution C

MaterialAmountFunction
Hematoxylin0.5 gDye
Distilled water100 mLSolvent

Solution D

MaterialAmountFunction
Ferric ammonium sulfate5 gMordant
Distilled water100 mLSolvent

Compounding procedure

Make each solution seperately.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into solution A for 24 hours.
  3. Wash with running tap water overnight.
  4. Place into solution B for 3-6 hours.
  5. Rinse with distilled water.
  6. Place into solution C overnight.
  7. Place into solution D until differentiated.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei – dense black

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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Reddy’s Alum Hematoxylin

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

Reddy's Alum Hematoxylin

8
steps
5
materials
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Materials

MaterialAmountFunction
Hematoxylin6.4 gDye
Ammonium alum60 gMordant
Ethanol, absolute200 mLSolvent
Glycerol160 mLStabiliser
Distilled water640 mLSolvent

Compounding Procedure

  1. Combine all the ingredients.
  2. Mix for a day.
  3. Leave in the dark for at least a month to ripen.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 3-5 minutes.
  3. Rinse with water.
  4. Differentiate lightly with 1% hydrochloric acid in 70% ethanol.
  5. Rinse with water.
  6. Blue with 0.1% sodium bicarbonate.
  7. Wash with water.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei – blue

Safety Note

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

References

  1. Reddy, P. H., (2001)
    Neurological Sciences Institute
    Oregon Health and Science University
March 30, 2023
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Schmorl’s Alum Hematoxylin

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

Schmorl's Alum Hematoxylin

8
steps
4
materials
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Materials

MaterialAmountFunction
Hematoxylin5 gDye
Ammonium alum100 gMordant
Distilled water1 LSolvent
100% ethanol60 mLSolvent

Compounding Procedure

  1. Dissolve the hematoxylin in the ethanol.
  2. Dissolve the Alum in the water.
  3. Combine the solutions and mix well.
  4. Leave at room temperature to ripen.

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 hematoxylin content indicates this solution is likely a strong regressive type.
  • 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., (1954)
    Histopathologic technique and practical histochemistry Ed.2
    Blakiston, New York, USA.
March 30, 2023
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Sass’ Alum Hematoxylin Variants

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

Sass' Alum Hematoxylin Variants

8
steps
5
materials
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Materials

MaterialVariation IVariation IIFunction
Hematoxylin10 g1 gDye
Ammonium alumsaturated50 gMordant
Distilled water1 L1 LSolvent
Sodium iodate10 g1 gOxidant
Glacial acetic acid30 mL–Acidifier

Compounding Procedure

  1. Dissolve the Alum in the water.
  2. Add the hematoxylin.
  3. Add the other ingredients.
  4. Filter.
  5. The solutions 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

  • Both of these formulae use 1 gram of sodium iodate for each gram of hematoxylin. However, 1 gram of hematoxylin is oxidized by 0.2 grams of sodium iodate, so the solutions should be expected to have a relatively short life.
  • Due to it’s high dye content, the Var I formula is likely regressive.
  • The Var II formula is likely progressive.
  • 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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Scott’s Alum Hematoxylin

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

Scott's Alum Hematoxylin

10
steps
6
materials
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Materials

MaterialAmountFunction
Hematoxylin3 gDye
95% ethanol300 mLSolvent
Potassium alum21 gMordant
Distilled water300 mLSolvent
Glycerol300 mLStabiliser
Glacial acetic acid30 mLAcidifier

Compounding Procedure

  1. Dissolve the hematoxylin in a portion of the water
  2. Add the glycerol and mix well.
  3. Dissolve the Alum in the remaining water with heat.
  4. Combine the two solutions.
  5. Add the ethanol and the acetic acid, mixing each time
  6. Plug the container loosely with cotton wool.
  7. Ripen by leaving in a warm, sunlit place for several weeks.
  8. When sufficiently ripened, store tightly stoppered in a cool, dark place.
  9. The solution is stable for years.

Section Preparation

Scott recommended that tissues be fixed with formalin or one of many common fixative solutions, but that strongly acid fixatives should be avoided since they permit only nuclei to be stained. He specified that paraffin sections should be fixed to the slide without melting the paraffin and that albumin be used only if necessary.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Remove mercury pigment with the iodine-thiosulphate sequence if necessary.
  3. Rinse well with tap water then with distilled water.
  4. Rinse off the water with a little alcohol and wipe around the section.
  5. Drop a little of the hematoxylin solution onto each section and leave for 10 minutes.
  6. Rinse off the hematoxylin with a little alcohol then rinse thoroughly with distilled water.
  7. Blue with Scott’s tap water substitute for a few minutes.
  8. Rinse well with distilled water.
  9. Counterstain if desired.
  10. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • The fixatives Scott mentioned as suitable included:
    • Formalin
    • Alcohol (presumably ethanol)
    • Carnoy’s acetic alcohol
    • Gilson’s alcohol chloroform acetic
    • Mercuric chloride
    • Formal sublimate (mercuric chloride formalin)
    • Acid sublimate mixtures (acid mercuric chloride mixtures)
    • Potassium dichromate mercuric chloride mixtures (Helly, Zenker formal)
    • Potassium dichromate (Müller’s fluid)
    • Potassium dichromate formalin
  • The fixatives that he said should be avoided included:
    • Strongly acid fixatives such as Zenker acetic and Tellyesniczky
  • This is a modification of Ehrlich’s formula. It differs by having about half the hematoxylin and a fixed amount of alum. It should therefore stain somewhat more selectively than Ehrlich’s formula, but otherwise would be very similar.
  • Scott noted the desirability of chemical oxidation, but made no recommendation. Complete oxidation would require 0.6 to 0.75 grams sodium iodate and half oxidation would require 0.3 to 0.35 grams.
  • The procedure Scott used indicates it is intended to be a progressive stain with some background colouration.
  • Although Scott recommended his tap water substitute for blueing, he noted that distilled water applied for long enough would also be effective, as would any other standard blueing solution.
  • Scott’s tap water substitute:
    MaterialAmount
    Potassium bicarbonate (KHCO3)2g
    Calcium chloride (CaCl2.2H2O)0.5-0.75g
    Magnesium sulphate (MgSO4.7H2O)20g
    Distilled water1L

Safety Note

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

References

  1. Scott, S. G. (1912)
    On successive double staining for histological purposes
    Journal of Pathology and Bacteriology, v. 16, p. 390-398.
March 30, 2023
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Seidelin’s Iron Hematoxylin

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

Seidelin's Iron Hematoxylin

6
steps
5
materials
print

Materials

Solution A

MaterialAmountFunction
Ferric chloride0.6 gMordant
Distilled water95 mLSolvent
Hydrochloric acid0.75 mLAcidifier

Solution B

MaterialAmountFunction
Hematoxylin1 gDye
100% ethanol100 mLSolvent

Compounding Procedure

  1. Make each solution separately.
  2. For use, combine 3 parts solution A with 2 parts 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.
  • This is a modification of Weigert’s iron hematoxylin.
  • The staining time should be determined by trial, but is likely about 10-20 minutes.

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:
    Seidelin, (1911). Parasitology, v. 4, p. 94.
March 30, 2023
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Slidders’ Alum Hematoxylin

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

Slidders' Alum Hematoxylin

6
steps
5
materials
print

This formulation differs from most others in that it is made from three stock solutions. It contains no added acid, relying on the high alum:dye ratio for it’s progressive staining. Consequently, the carry over of tap water fairly rapidly causes both dilution and raises the pH. Due to these, it has a shorter working life than many other formulations. It was designed to be quickly prepared in laboratories that stain large numbers of slides and prefer to use fresh solution daily, but which would like to avoid the poor background staining obtained with formulations that incorporate an acid to accentuate nuclei.

Materials

Stock mordant

MaterialAmountFunction
Potassium alum2 kgMordant
Distilled water19.8 LSolvent
Strong formalin200 mLPreservative

Stock hematoxylin

MaterialAmountFunction
Hematoxylin20 gDye
Strong formalin200 mLSolvent

Stock oxidizer

MaterialAmountFunction
Sodium iodate4 gOxidant
Distilled water200 mLSolvent

Compounding Procedure

Stock mordant

  1. Dissolve the Alum into 4 litres of distilled water, using heat.
  2. Add the balance of the water and the formalin.

Stock hematoxylin and stock oxidizer

  1. Dissolve each constituent into the appropriate solvent.

Working solution

  1. Place 400 mL of the stock mordant into a 2 litre flask.
  2. Add 20 mL stock hematoxylin and 20 mL stock oxidizer.
  3. Under a fume hood, bring to the boil, then remove immediately.
  4. Add 1600 mL stock mordant.
  5. The solution may be used immediately.

The formulated solution is stable for about 2 months at room temperature. The stock solutions are stable at least 6 months at room temperature.

The solution may also be made in smaller volumes according to a more traditional regimen:

MaterialAmountFunction
Hematoxylin1 gDye
Potassium alum100 gMordant
Distilled water1 LSolvent
Sodium iodate0.2 gOxidant

Compounding Procedure

  1. Combine the ingredients.
  2. Bring to the boil and immediately remove from heat.
  3. Cool to room temperature.
  4. The solution may be used immediately after it has cooled.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for about 5 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 formalin used as the solvent in the stock Alum and hematoxylin solutions should be concentrated, i.e. 37-40% aqueous formaldehyde. It acts as both a preservative and an anti-oxidant.
  • 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. Slidders, W. (1988)
    Preparation of Alum hematoxylin.
    Medical Laboratory Sciences, v 45, pp. 405.
March 30, 2023
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Unna’s Alum Hematoxylin

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

Unna's Alum Hematoxylin

8
steps
5
materials
print

Materials

MaterialAmountFunction
Hematoxylin3 gDye
Ammonium alum300 gMordant
Distilled water600 mLSolvent
100% ethanol300 mLSolvent
Sublimed sulfur6 gStabiliser

Compounding Procedure

  1. Dissolve the hematoxylin in the alcohol.
  2. Dissolve the Alum in the water.
  3. Combine the two solutions.
  4. Leave the solution at room temperature to ripen (days).
  5. Add the sulfur and mix well.

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 sulfur is added after the solution has properly ripened.
  • Unna said that the sulfur stabilized the solution in the oxidized state for some time. Others considered glycerol to be superior for this purpose.
  • Ammonium Alum dissolves in water at the rate of about one gram in 7 mL, and it is almost insoluble in ethanol. The formula should therefore require about 90 grams, so the amount specified is in considerable excess.
  • 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.
    Citing:
    Unna, (1892)
    Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik, v.8, p.438
    Leipzig
March 30, 2023
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Watson’s Alum Hematoxylin

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

Watson's Alum Hematoxylin

8
steps
7
materials
print

Materials

MaterialAmountFunction
Hematoxylin6 gDye
Ammonium alum6 gMordant
Distilled water300 mLSolvent
100% ethanol300 mLSolvent
Glycerol300 mLStabiliser
Glacial acetic acid30 mLAcidifier
Potassium permanganate or

Chloramine T or

Barium hydroxide

0.3 g

3.9 g

6 g

Oxidants

Compounding Procedure

  1. Dissolve the Alum in the water.
  2. If using potassium permanganate, add to the Alum solution.
  3. Dissolve the hematoxylin in ethanol.
  4. Combine the two solutions.
  5. Add the glycerol.
  6. Add the acetic acid (30 mL, unless using barium hydroxide, then add 120 mL)
  7. If using chloramine T or barium hydroxide, add to the solution.

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

  • Use only one of the oxidants named.
  • This is a modification of Ehrlich’s hematoxylin, and is said to be as effective. It was designed to be used immediately after preparation, eliminating the months required for atmospheric oxidation.
  • Gray gives a similar formula for Watson’s hemalum with potassium permanganate, but specifies only 0.6 grams alum. This may be an error.
  • 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. Bolles Lee, A.. Edited by Gatenby, J.B. and Beams, H.W., (1950)
    The Microtomist’s Vade-Mecum. 11 ed.,
    Churchill, London, UK.
    Citing:
    Watson, (1943)
    Journal of the Royal Microscopical Society, v. 63, p. 20
  2. 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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