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Protocols

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Gill’s Aluminum Hematoxylin Variants

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

Gill's Aluminum Hematoxylin Variants

6
steps
6
materials
print

Materials

MaterialSolution StrengthFunction
Single (I)Double (II)Triple (III)
Hematoxylin2 g4 g6 gDye
Aluminum sulphate17.6 g70.4 g158.4 gMordant
Distilled water750 mL750 mL750 mLSolvent
Ethylene glycol250 mL250 mL250 mLSolvent
Sodium iodate0.2 g0.4 g0.6 gOxidant
Glacial acetic acid20 mL20 mL20 mLAcidifier

Compounding procedures

  1. Mix the ethylene glycol and water.
  2. Add the hematoxylin, then the sodium iodate.
  3. Add the aluminum sulphate and the acetic acid
  4. Stir for an hour at room temperature.
  5. Filter before use.
  6. The solutions may be used immediately, and are stable for approximately one year.

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 three formulations are for progressive staining.
  • The single strength solution is for cytology. Staining time is 2 minutes. The double strength solution is for paraffin sections. Staining time is 3 minutes. The triple strength solution is for paraffin sections. Staining time is 1.5 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.
  • The aluminum sulphate should be Al2(SO4)3.18 H2O. Adjust the amount if different.
  • Similarly, the hematoxylin should be anhydrous. If hydrated (C16H14O6.3 H2O) use 2.36 g, 4.72 g and 7.08 g respectively.

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.
March 30, 2023
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Gluck’s Impregnation for Reticulin on Zenker Fixed Tissue

By Metal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type
Protocol

Gluck's Impregnation

for Reticulin on Zenker Fixed Tissue

20
steps
12
materials
print

Materials

  • Oxalic acid, 5% aqu.
  • Silver nitrate, 10% aqu.
  • Strong ammonium hydroxide (s.g. 0.88).
  • Sodium hydroxide, 40% aqu.
  • Formalin, 5% aqu.
  • Yellow gold chloride, 0.2% aqu.
  • Neutral red, 1% aqu.
  • Gram’s iodine.
    MaterialAmount
    Iodine1g
    Potassium iodide2g
    Distilled water300mL

    Mix the iodine and potassium iodide in a 500 mL flask. Add 5 mL of the water. When the iodine has dissolved, make up to 300 mL with distilled water.

  • Cajal’s Solution
    MaterialAmount
    Strong formalin15mL
    Ammonium bromide2g
    Water85mL

Preparation of Gluck’s Ammoniacal Silver

  1. Place 10 mL of 10% silver nitrate in a flask.
  2. Add 0.5 mL of 40% sodium hydroxide.
  3. Allow to settle, then decant the supernatent.
  4. Wash the precipitate, allow to settle, then decant a few times.
  5. While swirling, slowly add drops of strong ammonium hydroxide until the precipitate just redissolves.
  6. Dilute to 100 mL with distilled water, then add 2 mL pyridine.

Tissue Sample

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

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Rinse well with tap water.
  3. Place in Gram’s iodine for 4-8 hours.
  4. Rinsewith 70% ethanol.
  5. Rinse well with tap water.
  6. Bleach in 5% sodium thiosulphate for 5 minutes.
  7. Wash well with tap water.
  8. Place in Cajal’s solution at 37°C for 24 hours.
  9. Wash with tap water.
  10. Place in Gluck’s ammoniacal silver for 5 minutes.
  11. Rinse with distilled water.
  12. Place in 5% formalin for 5 minutes.
  13. Wash with tap water.
  14. Tone with 0.2% gold chloride solution until grey.
  15. Rinse with distilled water.
  16. Place in 5% sodium thiosulphate for 5 minutes.
  17. Wash well with tap water.
  18. Counterstain with neutral red for 1 minute.
  19. Rinse with tap water.
  20. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Nuclei  –  red
  • Background  –  grey

Notes

  • Ensure that both the ammonium hydroxide and sodium hydroxide are fresh and full strength. Keep both well stoppered when not in use. For the ammonium hydroxide, pour sufficient for use from the stock bottle into a beaker, then immediately restopper the stock bottle. Do not return excess ammonium hydroxide to the stock bottle.
  • After making the ammoniacal silver solution but before adding the pyridine, smell the solution to ensure it has only a faint smell of ammonia. If the smell of ammonia is strong it indicates that too much ammonium hydroxide has been added. If so, it is preferable to make the solution again. Improperly made ammoniacal silver solutions can affect the quality of the impregnation.
  • The formalin used to make Cajal’s solution should be neutralised, but do not use buffered formalin. Neutral formalin in this context may be made by keeping strong formalin over marble chips. However, be very careful as the gas given off may increase the pressure inside the container and cause an explosion. Either apply a cap loosely so gas can escape, or use a fermentation lock.
  • Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black reticulin fibres on a pale grey-brown background. Others tone longer (a few minutes) to produce black reticulin fibres on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.

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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Goldman’s Iron Hematoxylin

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

Goldman's Iron Hematoxylin

10
steps
8
materials
print

Materials

Solution A

MaterialAmountFunction
Ferric ammonium sulfate4 gMordant
Distilled water100 mLSolvent
Acetic acid, glacial1 mLAcidifier
Sulfuric acid0.12 mLAcidifier

Solution B

MaterialAmountFunction
Picric acid, saturated aqueous100 mLDye and acid
Sulfuric acid0.1 mLAcidifier

Solution C

MaterialAmountFunction
Hematoxylin0.5 gDye
Distilled water100 mLSolvent

Solution D

MaterialAmountFunction
70% ethanol100 mLSolvent
Lithium carbonate, saturated aqueous5 dropsBase

Compounding procedures

  1. Make each solution separately.
  2. The aqueous hematoxylin should be ripened before use.

Protocol

  1. Bring sections to water, removing mercury pigment if necessary.
  2. Place into solution A for 30 minutes to 24 hours.
  3. Wash in running tap water for 10 minutes.
  4. Place in solution B for 3 hours or longer.
  5. Wash in running tap water for 15 minutes.
  6. Place in solution C for 1 hour.
  7. Wash in running tap water for 15 minutes.
  8. Blue with solution D.
  9. Counterstain if desired.
  10. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Basophil cytoplasm  –  grey
  • Background  –  as counterstain or unstained

Notes

  • The stock solutions are stable for some time.
  • The technique was originally intended for the demonstration of protozoa.
  • The method was designed for paraffin sections of material fixed with formalin variants, Bouin’s or Zenker’s fluids.
  • Overstaining occurs only if sections are left in hematoxylin for several hours.
  • Bouin fixed tissue is not as intensely stained as with other fixatives.
  • The time in picric acid (solution B) is necessary, reducing it causes overstaining.

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.
    Citing:
    Goldman, (1951)
    American Journal of Clinical Pathology,
    v.21, p.198
March 30, 2023
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Goldner’s Trichrome for Muscle and Collagen

By Protocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step
Protocol

Goldner's Trichrome

for Muscle and Collagen

11
steps
9
materials
print

Materials

  • Weigert’s iron hematoxylin or equivalent
  • Solution A
    MaterialVariation 1Variation 2Variation 3
    Ponceau 2R0.7g0.75g–
    Acid fuchsin0.3g0.25g–
    Azophloxine–0.1g5g
    Acetic acid, glacial2mL2mL2mL
    Distilled water1L1L1L
  • Solution B
    MaterialAmount
    Acetic acid, glacial10mL
    Distilled water1L
  • Solution C
    MaterialAmount
    Orange G20g
    Phosphomolybdic acid40g
    Distilled water1L
  • Solution D
    MaterialAmount
    Light green SF2g
    Acetic acid, glacial2mL
    Distilled water1L

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation.


Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with Weigert’s iron hematoxylin, or equivalent.
  3. Place into one of the variants of solution A for 5 minutes.
  4. Rinse with solution B.
  5. Place into solution C until collagen is decolorised.
  6. Rinse with solution B.
  7. Place into solution D for 5 minutes.
  8. Place into solution B for 5 minutes.
  9. Blot.
  10. Dehydrate rapidly with absolute ethanol.
  11. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Erythrocytes  –  orange
  • Cytoplasm  –  red
  • Collagen  –  green

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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Gridley’s Impregnation for Reticulin

By Metal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type
Protocol

Gridley's Impregnation

for Reticulin

17
steps
9
materials
print

Materials

  • Silver nitrate, 2% aqu.
  • Silver nitrate, 20% aqu.
  • Strong ammonium hydroxide (s.g. 0.88).
  • Sodium hydroxide, 40% aqu.
  • Periodic acid, 0.5% aqu.
  • Formalin, 3% aqu.
  • Yellow gold chloride, 0.5% aqu.
  • Sodium thiosulphate, 5% aqu.
  • Neutral red, 1% aqu.

Preparation of Da Fano’s Ammoniacal Silver

  1. Place 10 mL of 20% silver nitrate in a flask.
  2. Add 0.2 mL of 40% sodium hydroxide.
  3. While swirling, slowly add drops of strong ammonium hydroxide until the precipitate just redissolves.
  4. Make up to 80 mL with distilled water.

Tissue Sample

6 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Oxidise with 0.5% periodic acid for 15 minutes.
  3. Rinse well with distilled water.
  4. Sensitise with 2% silver nitrate for 30 minutes.
  5. Rinse well with distilled water.
  6. Treat with da Fano’s ammoniacal silver for 15 minutes.
  7. Rinse with distilled water.
  8. Reduce in 3% formalin for 3 minutes.
  9. Rinse well with tap water.
  10. Rinse with distilled water.
  11. Tone with 0.5% gold chloride solution for 5 minutes.
  12. Rinse with distilled water.
  13. Fix in 5% sodium thiosulphate for 5 minutes.
  14. Wash well with running tap water.
  15. Counterstain with neutral red for 1 minute.
  16. Rinse with tap water.
  17. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Nuclei  –  red
  • Background  –  grey

Notes

  • Ensure that both the ammonium hydroxide and potassium hydroxide are fresh and full strength. Keep both well stoppered when not in use. For the ammonium hydroxide, pour sufficient for use from the stock bottle into a beaker, then immediately restopper the stock bottle. Do not return excess ammonium hydroxide to the stock bottle.
  • After making the ammoniacal silver solution, smell the solution to ensure it has only a faint smell of ammonia. If the smell of ammonia is strong it indicates that too much ammonium hydroxide has been added. If so, it is preferable to make the solution again. Improperly made ammoniacal silver solutions can affect the quality of the impregnation.
  • 3% formalin is made by diluting strong formalin with tap water (3 mL strong formalin, 97 mL tap water).
  • Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Although the method specifies 5 minutes toning in 0.5% gold chloride, this is not mandatory. Many microscopists prefer to tone for about 15 seconds to produce brown-black reticulin fibres on a pale grey-brown background. Others tone longer (a few minutes) to produce black reticulin fibres on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.
  • The use of periodic acid followed by an easily reducible silver solution is obviously similar to the Jones’ method for basement membranes and carbohydrates. Note, however, that Gridley’s method applies ammoniacal silver solution at room temperature after sensitising in aqueous silver nitrate at room temperature, then applies formalin as a reducing agent, i.e. it is an argyrophil reaction, while the Jones’ method applies a methenamine silver solution at 56°C for a longer time, without sensitising, and does not use an external reducer, i.e. it is an induced argentaffin reaction. Clearly, in Gridley’s method the periodic acid is being used for the same purpose as the potassium permanganate of the Mallory bleach in other methods.

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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Gordon & Sweets Impregnation for Reticulin

By Metal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type
Protocol

Gordon & Sweets Impregnation

for Reticulin

19
steps
11
materials
print

Materials

  • Silver nitrate, 10% aqu.
  • Strong ammonium hydroxide (s.g. 0.880).
  • Sodium hydroxide, 3% aqu.
  • Oxalic acid, 1% aqu.
  • Iron alum, 2.5% aqu.
  • Formalin, 10% aqu.
  • Yellow gold chloride, 0.2% aqu.
  • Sodium thiosulphate, 3% aqu.
  • Neutral red, 1% aqu.
  • Mallory bleach
    MaterialAmount
    Potassium permanganate, 0.5% aqu.47.5mL
    Sulphuric acid, 3% aqueous2.5mL

Preparation of Ammoniacal Silver

  1. Place 5 mL of the 10% silver nitrate in a 100 mL flask.
  2. Using a pasteur pipette, add a drop of strong ammonium hydroxide then swirl the solution for a few seconds.
  3. A precipitate will form at first.
  4. Continue adding ammonium hydroxide drop by drop and swirling until the precipitate is just redissolved.
  5. Add 5 mL of 3% sodium hydroxide and mix well.
  6. A precipitate will again form.
  7. Add drops of ammonium hydroxide until the precipitate is just redissolved, leaving a faint opalescence.
  8. Dilute to 50 mL with distilled water.

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Oxidise with the Mallory bleach for 5 minutes.
  3. Bleach in Oxalic acid for a few minutes.
  4. Rinse with distilled water.
  5. Rinse well with tap water.
  6. Sensitise with iron alum solution for 15 minutes.
  7. Rinse well with distilled water.
  8. Treat with ammoniacal silver for 30 seconds.
  9. Rinse well with distilled water.
  10. Reduce in formalin for 1 minute.
  11. Rinse well with tap water.
  12. Rinse with distilled water.
  13. Tone with 0.2% gold chloride solution.
  14. Rinse with distilled water.
  15. Fix in sodium thiosulphate for 10 minutes.
  16. Wash well with running tap water.
  17. Counterstain with neutral red for 1 minute.
  18. Rinse with tap water.
  19. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Nuclei  –  red
  • Background  –  grey

Notes

  • Gordon & Sweets also suggested a variant that used one of Foot’s ammoniacal silver solutions. Apart from that, the method is the same as given above.

    Foot’s ammoniacal silver

    • To 10 mL of 1% silver nitrate, add 0.1 mL of 40% potassium hydroxide.
    • Add strong ammonium hydroxide by drops until the precipitate just redissolves.
    • Make up the volume to 100mL with distilled water.
  • Ensure that both the ammonium hydroxide and sodium hydroxide are fresh and full strength. Keep both well stoppered when not in use. For the ammonium hydroxide, pour sufficient for use from the stock bottle into a beaker, then immediately restopper the stock bottle. Do not return excess ammonium hydroxide to the stock bottle.
  • After making the ammoniacal silver solution, smell the solution to ensure it has only a faint smell of ammonia. If the smell of ammonia is strong it indicates that too much ammonium hydroxide has been added. If so, it is preferable to make the solution again. Improperly made ammoniacal silver solutions can affect the quality of the impregnation.
  • Most references to the Gordon & Sweets’ reticulin stain specify that the ammoniacal silver solution should be made with 10.2% aqueous silver nitrate and 3.1% aqueous sodium hydroxide. No explanation is given. The 10% and 3% solutions respectively, as given by Gray, work satisfactorily.
  • Iron alum is ferric ammonium sulphate. For routine formalin fixed tissue 15 minutes in the iron alum is usually sufficient. If necessary the time may be extended up to 2 hours.
  • 10% formalin is made by diluting strong formalin 1:10 with tap water (10 mL strong formalin, 90 mL tap water).
  • Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black reticulin fibres on a pale grey-brown background. Others tone longer (a few minutes) to produce black reticulin fibres on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.

Safety Note

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

References

  1. Drury, R A, and Wallington, E A, (1967).
    Carleton’s histological technique., Ed. 5.
    Oxford University Press, London, England.
  2. Culling, C F A, Allison, R T, Barr, W T, (1985).
    Cellular pathology technique., Ed. 4.
    Butterworths, London, England.
  3. 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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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
print

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
print

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
print

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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