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Régaud’s Iron Hematoxylin

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

Régaud's Iron Hematoxylin

9
steps
7
materials
print

Materials

Solution A

MaterialAmountFunction
Ferric ammonium sulfate5 gMordant
Distilled water100 mLSolvent

Solution B

MaterialAmountFunction
Hematoxylin1 gDye
90% ethanol10 mLSolvent
Distilled water80 mLSolvent
Glycerol10 mLStabiliser

Solution C

MaterialAmountFunction
Potassium ferricyanide2.5 gBleach
Sodium borate2 gpH control
Distilled water100 mLSolvent

Compounding Procedure

  1. Make each solution separately.
  2. Solutions A and B may be reused provided they are not contaminated by each other.
  3. Solution C should not be reused.

Protocol

  1. Bring sections to distilled water with xylene and ethanol.
  2. Place into solution A at 50°C for 30 minutes.
  3. Rinse well with distilled water.
  4. Place into solution B at 50°C for 30 minutes.
  5. Rinse well with distilled water.
  6. Place into solution C until differentiated.
  7. Wash in running tap water until blue.
  8. Counterstain as required.
  9. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Background  –  as counterstain or unstained

Notes

  • The solutions are stable for some time.
  • Solution C is Weigert’s ferricyanide bleach and should be used fresh.

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
Love0

Robb-Smith’s Impregnation for Reticulin

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

Robb-Smith's Impregnation

for Reticulin

20
steps
10
materials
print

Materials

  • Silver nitrate, 5% aqu.
  • Silver nitrate, 10% aqu.
  • Sodium hydroxide, 10% aqu.
  • Strong ammonium hydroxide (s.g. 0.88)
  • Ammonium hydroxide 10% aqu.
  • Potassium permanganate, 0.25% aqu.
  • Oxalic acid, 1.5% aqu.
  • Formalin, 37.5% aqu.
  • Yellow gold chloride, 0.2% aqu.
  • Sodium thiosulphate, 5% aqu.

Preparation of Robb-Smith’s Ammoniacal Silver

  1. Place 28.5 mL of 10% aqueous silver nitrate in a flask.
  2. Add 0.6 mL of 10% aqueous sodium hydroxide
  3. Add strong ammonium hydroxide by drops until the precipitate just dissolves.
  4. The solution should have a faint opalescence.
  5. Dilute to 100 mL with distilled water.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue mounted on slides are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended. The paraffin wax does not need to be removed.

Protocol

  1. Place sections with the paraffin wax intact in 10% ammonia for 15 minutes
  2. Wash with tap water.
  3. Oxidize with 0.25% potassium permanganate for 5 minutes.
  4. Wash with tap water.
  5. Place in oxalic acid until bleached.
  6. Wash with tap water.
  7. Place in 5% silver nitrate for 1 hour.
  8. Rinse with distilled water.
  9. Treat with ammoniacal silver solution for 15 minutes.
  10. Wash with distilled water.
  11. Place in 37.5% formalin for 3 minutes.
  12. Wash with tap water.
  13. Rinse with distilled water.
  14. Tone with 0.2% gold chloride solution for 3 minutes.
  15. Rinse with distilled water.
  16. Fix in 5% sodium thiosulphate for 1 minute.
  17. Wash well with running tap water.
  18. Dehydrate with ethanol.
  19. Remove paraffin wax and clear with a few changes of xylene.
  20. Mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Background  –  grey

Notes

  • The 10% ammonium hydroxide is made by diluting 1 part strong ammonium hydroxide with 9 parts distilled water.
  • The 37.5% formalin solution (15% formaldehyde) is made by diluting 37.5 mL strong formalin with 62.5 mL tap water.
  • Ensure that the strong ammonium hydroxide is fresh and full strength. Keep well stoppered when not in use. Pour sufficient for use into a beaker, then immediately restopper the bottle. Do not return unused solution to the stock bottle.
  • Improperly made ammoniacal silver solutions can affect the quality of the impregnation. There should be a faint, persistent opalescence, with only a faint smell of ammonia.
  • 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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Studricka’s Impregnation for Reticulin in Decalcified Tissue

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

Studricka's Impregnation

for Reticulin in Decalcified Tissue

12
steps
7
materials
print

Materials

  • Silver nitrate, 10% aqu.
  • Silver nitrate, 3% aqu.
  • Strong ammonium hydroxide (s.g. 0.88)
  • Sodium hydroxide, 10% aqu.
  • Formalin, 10% aqu.
  • Yellow gold chloride, 0.5% aqu.
  • Sodium thiosulphate, 5% aqu.

Preparation of Studricka’s Ammoniacal Silver

  1. Place 100 mL of 10% aqueous silver nitrate in a flask.
  2. Add 10% aqueous sodium hydroxide until no more precipitate forms (with a Pasteur pipette).
  3. Add strong ammonium hydroxide by drops until the precipitate just dissolves.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed and acid decalcified 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. Place in 3% silver nitrate for 4 days.
  3. Wash with distilled water.
  4. Place in ammoniacal silver solution until sections are yellow-brown.
  5. Rinse with distilled water.
  6. Place in 10 formalin until sections are dark brown.
  7. Wash with distilled water.
  8. Tone with 0.5% gold chloride until sections are grey-black.
  9. Wash with distilled water.
  10. Fix in 5% sodium thiosulphate for 1 hour.
  11. Wash well with running tap water.
  12. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Background  –  grey

Notes

  • Ensure that the strong ammonium hydroxide is fresh and full strength. Keep well stoppered when not in use. Pour sufficient for use into a beaker, then immediately restopper the bottle. Do not return unused solution to the stock bottle.
  • Improperly made ammoniacal silver solutions can affect the quality of the impregnation. There should be a faint, persistent opalescence, with only a faint smell of ammonia.
  • 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.
  • Gray notes that Zimmerman recommended treatment with 3% silver nitrate for 2 days (step 2), and that the ammoniacal silver solution should be diluted 1 part with 3 parts distilled water (step 4).

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
Love0

Wilder’s Impregnation for Reticulin

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

Wilder's Impregnation

for Reticulin

19
steps
12
materials
print

Materials

  • Silver nitrate, 2% aqu.
  • Strong ammonium hydroxide (s.g. 0.88)
  • Sodium hydroxide, 40% aqu.
  • Potassium permanganate, 1% aqu.
  • Oxalic acid, 5% aqu.
  • Uranium nitrate, 1% aqu.
  • Yellow gold chloride, 0.2% aqu.
  • Sodium thiosulphate, 5% aqu.
  • Neutral red, 1% aqu.
  • Developer
    MaterialAmount
    Strong formalin5mL
    Uranium nitrate0.15g
    Water1L

Preparation of Bielchowsky’s Ammoniacal Silver

  1. Place 48 mL of 2% silver nitrate in a flask.
  2. Add 0.4 mL of 40% sodium hydroxide.
  3. While swirling, slowly add drops of strong ammonium hydroxide until the precipitate just redissolves.
  4. Make up to 100 mL with distilled water.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory, but see the notes for comments about Zenker and Helly type fixed tissue. A section adhesive is recommended.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Oxidize with 1% potassium permanganate for 1 minute.
  3. Rinse well with tap water.
  4. Bleach in Oxalic acid for 1 minute.
  5. Rinse well with tap water.
  6. Sensitize with 1% uranium nitrate solution for 5-10 seconds.
  7. Rinse with distilled water.
  8. Treat with Bielchowsky’s ammoniacal silver for 1 minute.
  9. Rinse briefly with 90% ethanol.
  10. Place in the developer 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 5% sodium thiosulphate for 1 minute.
  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

  • For Zenker and Helly type fixatives, Wilder recommended a slightly different procedure.
    • Replace the Mallory bleach (steps 2-4) with 10% phosphomolybdic acid treatment for 1 minute, followed by a water rinse.
    • Use Foot’s ammoniacal silver solution instead of Bielchowsky’s.

      Foot’s ammoniacal silver: To 10 mL of 1% silver nitrate in a flask, add 0.1 mL of 40% potassium hydroxide. Add strong ammonium hydroxide drop by drop while shaking the solution until the precipitate just dissolves. Make up to 100 mL 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.
  • It is sometimes difficult to obtain uranium nitrate, particularly if it requires international transportation.
  • The strong formalin used to make the developer should be neutralized, 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
Love0

Zimmerman’s Impregnation for Reticulin in Decalcified Tissue

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

Zimmerman's Impregnation

for Reticulin in Decalcified Tissue

12
steps
7
materials
print

Materials

  • Silver nitrate, 10% aqu.
  • Silver nitrate, 3% aqu.
  • Strong ammonium hydroxide (s.g. 0.88)
  • Sodium hydroxide, 10% aqu.
  • Formalin, 10% aqu.
  • Yellow gold chloride, 0.5% aqu.
  • Sodium thiosulphate, 5% aqu.

Preparation of Zimmerman’s Ammoniacal Silver

  1. Place 25 mL of 10% aqueous silver nitrate in a flask.
  2. Add 10% aqueous sodium hydroxide until no more precipitate forms (with a Pasteur pipette).
  3. Add strong ammonium hydroxide by drops until the precipitate just dissolves.
  4. Dilute to 100 mL with distilled water.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed and acid decalcified 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. Place in 3% silver nitrate for 2 days.
  3. Wash with distilled water.
  4. Place in ammoniacal silver solution until sections are yellow-brown.
  5. Rinse with distilled water.
  6. Place in 10 formalin until sections are dark brown.
  7. Wash with distilled water.
  8. Tone with 0.5% gold chloride until sections are grey-black.
  9. Wash with distilled water.
  10. Fix in 5% sodium thiosulphate for 1 hour.
  11. Wash well with running tap water.
  12. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Background  –  grey

Notes

  • Ensure that the strong ammonium hydroxide is fresh and full strength. Keep well stoppered when not in use. Pour sufficient for use into a beaker, then immediately restopper the bottle. Do not return unused solution to the stock bottle.
  • Improperly made ammoniacal silver solutions can affect the quality of the impregnation. There should be a faint, persistent opalescence, with only a faint smell of ammonia.
  • 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.
  • This method is a modification of Studricka’s technique using a shorter time in silver nitrate (step 2), and a more dilute ammoniacal silver solution (step 4).

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
Love0

Rozas’ Iron Hematoxylin

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

Rozas' Iron Hematoxylin

7
steps
8
materials
print

Rozas’ iron hematoxylin is of interest as it contains aluminum and iron salts in one solution. Both are mordants used with hematoxylin, and there will presumably be some kind of competition for available binding sites which could modify the solution’s staining characteristics. Since iron can be used to displace aluminum as a mordant for hematoxylin, it is likely that the solution stains more as an iron hematoxylin than as an aluminum hematoxylin.

Materials

Solution A

MaterialAmountFunction
Hematoxylin0.6 gDye
Ferric ammonium sulfate1 gMordant
Aluminum chloride1.2 gMordant
Distilled water74 mLSolvent
95% ethanol6 mLSolvent
Glycerol20 mLStabiliser

Solution B

MaterialAmountFunction
Ferric ammonium sulfate20 gMordant
Distilled water100 mLSolvent

Compounding Procedure

  1. For solution A, dissolve the hematoxylin in the ethanol.
  2. Dissolve the ferric ammonium sulfate and the aluminum chloride in the water.
  3. Combine, then add the glycerol.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into solution A for 12-24 hours.
  3. Place into solution B until differentiated.
  4. Wash well in running tap water to blue.
  5. Rinse with distilled water.
  6. Counterstain if desired.
  7. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Background  –  as counterstain or unstained

Notes

  • The presence of glycerol indicates that solution A may be stable for some time.
  • The staining procedure indicates that the technique is intended as a primary stain. It is likely a modification of Heidenhain’s method and would demonstrate much the same material.

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:
    Rozas, (1935)
    Zietschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik,
    v.52, p.1. Leipzig.
March 30, 2023
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Mallory’s Alum Hematoxylin

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

Mallory's Alum Hematoxylin

8
steps
4
materials
print

Materials

MaterialAmountFunction
Hematoxylin2.5 gDye
Potassium alum50 gMordant
Distilled water1 LSolvent
Thymol2.5 gPreservative

Compounding Procedure

  1. Dissolve the dye and Alum in the water.
  2. Add the thymol.
  3. Allow to ripen before use.

    4. </ol

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 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
Love0

Mallory & Wright’s Alum Hematoxylin Variants

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

Mallory & Wright's Alum Hematoxylin Variants

8
steps
3
materials
print

Materials

MaterialStandard FormulaStrong FormulaFunction
Hematoxylin1 g1 gDye
Ammonium alum, saturated aqueous100 mL100 mLMordant
Distilled water300 mL–Solvent

Compounding Procedure

  1. Dissolve the hematoxylin in the Alum solution by warming if necessary.
  2. Add the water if the standard solution is being used.
  3. Plug the container with cotton wool.
  4. Ripen for approximately ten days.
  5. Place the stopper tightly.
  6. Filter before use. The solution is stable for 2-3 months.
  7. A small crystal of thymol may be added.

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 standard solution is for routine, formalin fixed tissues.
  • The strong solution is recommended for Zenker fixed tissues.
  • 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. Mallory, F. B. & Wright, J.H., (1904)
    Pathological technique, Ed.3
    W. B. Saunders, Philadelphia, USA.
March 30, 2023
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Mallory’s PTAH

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

Mallory's PTAH

6
steps
3
materials
print

Materials

MaterialAmountFunction
Hematein,1 g.Dye
Phosphotungstic acid10 gMordant
Distilled water1 LSolvent

Compounding Procedure

  1. Dissolve the hematein in 200 mL of the water and the phosphotungstic acid in the rest.
  2. Combine the solutions.
  3. Keep in a bottle with a tightly fitting cap to ensure atmospheric oxygen is excluded.
  4. The stain may be used after a day or two.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Perform a Mallory bleach using 0.25% potassium permanganate.
  3. Rinse well with water.
  4. Place into the PTAH solution for 12 – 24 hours at room temperature.
  5. Rinse well with water.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei, erythrocytes, fresh fibrin, muscle striations  –  blue
  • Background  –  red

Notes

  • If formalin fixed, the sections may be treated with acid dichromate for 30 minutes.
    MaterialAmount
    Potassium dichromate, 3% aqueous36mL
    10% hydrochloric acid in absolute ethanol.12mL
  • The staining may be done in an oven at about 60°C for a few hours, but the results are often less satisfactory.
  • Many substances have been stated to be stained blue. Sometimes the red staining overshadows blue staining, and the section may need to be washed with water to remove excess red. Excess blue may be removed by extending the treatment with ethanol during dehydration.
  • PTAH may also be made with hematoxylin and oxidized as any other hematoxylin solution. Atmospheric oxidation is often recommended, but takes a few months. Place the solution in a flask with a loose cotton batting stopper to facilitate exposure to air. Test periodically and, when staining is satisfactory, place into a bottle with a tightly fitting lid to inhibit atmospheric oxidation.
  • Alternatively, the hematoxylin may be chemically oxidized. This is usually done with 12 mL of 1% aqueous potassium permanganate. The solution may be used after a day or two. Sodium iodate may also be used. Half the hematoxylin will be oxidized with 0.1 grams sodium iodate. If brought to a boil and allowed to cool, it may be used immediately, or it may be allowed to oxidize at room temperature for a few days.

Safety Note

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

References

  1. Bancroft, J. D. and Stevens, A.,
    Theory and Practice of Histological Techniques, Ed. 2,
    Churchill Livingstone, London, UK.
March 30, 2023
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Mann’s Alum Hematoxylin

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

Mann's Alum Hematoxylin

8
steps
6
materials
print

Materials

MaterialAmountFunction
Hematein6 gDye
Potassium alum35 gMordant
Distilled water350 mLSolvent
95% ethanol320 mLSolvent
Glycerol250 mLStabilizer
Glacial acetic acid30 mLAcidifier

Compounding Procedure

  1. Dissolve the dye in the acetic acid.
  2. Mix the ethanol and glycerol together, and add to the dye in acetic acid.
  3. Dissolve the Alum in the water, and add to the dye solution.
  4. 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.
  • The high dye content indicates 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. 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
Love0