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Proescher & Arkush Iron Alum-Celestine Blue for Nuclei

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

Proescher & Arkush Iron Alum-Celestine Blue

for Nuclei

6
steps
4
materials
print

Materials

  • An eosin solution, or other counterstain
  • Iron alum-celestine blue
    MaterialAmount
    Ferric ammonium sulphate5g
    Celestine blue B0.5g
    Distilled water100mL

    Dissolve the ferric ammonium sulphate in the distilled water. Add the celestine blue B. Boil for 5 minutes. Cool and filter.

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 the iron alum-celestine blue solution for 3 minutes to 2 hours.
  3. Wash with tap water.
  4. Counterstain with eosin or other counterstain.
  5. Dehydrate with 95% and absolute ethanols.
  6. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Cytoplasm  –  pink

Notes

  • The iron alum-celestine blue solution is stable for a few months.
  • A staining time of 5-10 minutes is usually satisfactory for formalin fixed tissues.
  • This method has been recommended as a substitute for Hematoxylin and Eosin.
  • Proescher & Arkush also recommended the dyes gallamine blue and gallocyanin. Neither dye is as stable as celestine blue, although the color with gallocyanin most closely resembles alum hematoxylin of all three dyes.

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:
    Proescher and Arkush, (1928)
    Stain Technology, v. 3, pp. 36
March 30, 2023
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Akita & Kaneko’s Hemalum for Elastic Fibers

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

Akita & Kaneko's Hemalum

for Elastic Fibers

5
steps
6
materials
print

Materials

MaterialAmount
Hematoxylin100mg
Ethanol, 70%100mL
Sodium iodate20mg
Potassium alum300mg
Chloral hydrate5g
Citric acid100mg

Staining Solution Preparation

  1. Dissolve the hematoxylin into the ethanol.
  2. Add the potassium alum, then the other ingredients.
  3. Mix well for 15 to 20 minutes, then filter.
  4. At this point the solution is stable for 2 months at room temperature.
  5. Immediately prior to use, adjust to pH 8.0 with saturated aquueous lithium carbonate or 1N potassium hydroxide.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Helly and Bouin are also suitable. Other fixatives should be tested.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into freshly pH adjusted staining solution for 30-60 minutes.
  3. Wash with running tap water for 10 minutes.
  4. Dehydrate with ethanols.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  blue
  • Collagen  –  pale violet

Notes

  • Note that this is an ethanolic variant of Mayer’s hemalum.
  • If sodium iodate is left out, the staining solution lasts 3-4 days, but the staining time is extended to 60 minutes.
  • The solution may also be made by diluting 30 mL Mayer’s hemalum with 70 mL absolute ethanol, mixing for 20 minutes, and filtering before adjusting the pH. Staining requires 60 minutes and the results are paler.

Safety Note

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

References

  1. Akita, M. and Kaneko, K., (1981)
    An improved hematoxylin method with application of Mayer’s hemalum for staining of elastic fibres.
    Stain Technology, v. 56, p. 327
  2. Kaneko, K. and Akita, M., (1978)
    On the staining of elastic fibres with alum hematoxylin.
    Stain Technology, v. 53, p. 43.
March 30, 2023
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Gabe’s Aldehyde Fuchsin

By Aldehyde Fuchsin, Dye Type, Elastic Fibers, Protocols, Stain Target, Stain Type
Protocol

Gabe's Aldehyde Fuchsin

6
steps
9
materials
print

Materials

Stock powder

MaterialAmount
Pararosanilin5g
Paraldehyde5mL
Hydrochloric acid10mL
Distilled water1L

Dye Preparation Procedures

  1. Add the dye to the water and boil.
  2. Cool to room temperature.
  3. Add paraldehyde and hydrochloric acid.
  4. Ripen at RT two days. Filter. Wash precipitate with 50mL distilled water. Dry the ppt and paper at 60°C.
  5. Store in a labelled container. Stable for about ten years.

Working solution

MaterialAmount
Stock powder0.25g
Ethanol, 70%200mL
Acetic acid, glacial2mL

Acid ethanol

MaterialAmount
Hydrochloric acid, conc.1mL
Ethanol, 95%200mL

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 in the working solution for 5 minutes.
  3. Rinse well with running tap water.
  4. Rinse with acid ethanol for 20-30 seconds to remove excess dye.
  5. Counterstain if wished.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  purple
  • Mast cells  –  purple
  • Pituitary βcells  –  purple
  • Sulphated mucins  –  purple
  • Background  –  as the counterstain
  • Nuclei  –  as the nuclear stain

Notes

  • The basic fuchsin used for this solution should be one that is suitable for Schiff’s reagent, i.e., it should have a high pararosanilin content. Both methods involve forming a compound between an aldehyde and dye.
  • Light counterstaining with a progressive alum hematoxylin and eosin is also suitable.
  • Many other counterstains can be used, including methods such as Masson’s trichrome.

Safety Note

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

References

  1. Kiernan. J.A., (1999)
    Histological and histochemical methods: Theory and practice, Ed. 3
    Butterworth Heinemann, Oxford, UK.
    Citing:
    Gabe, M., (1976)
    Histological techniques.
    Masson, Paris.
March 30, 2023
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Gomori’s Aldehyde Fuchsin

By Aldehyde Fuchsin, Dye Type, Elastic Fibers, Intracytoplasmic Granules, Mast Cells, Protocols, Stain Target, Stain Type
Protocol

Gomori's Aldehyde Fuchsin

7
steps
4
materials
print

Materials

Solution A

MaterialAmount
Basic fuchsin1g
Paraldehyde, fresh1mL
Hydrochloric acid, conc.1mL
Ethanol, 70%200mL

Compounding Procedure

  1. Dissolve the dye in the ethanol.
  2. Add paraldehyde and hydrochloric acid.
  3. Ripen at room temperature for 48-72 hours.
  4. Refrigerate. The solution is stable for 2-3 months.

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. Wash with water.
  3. Rinse with 70% ethanol.
  4. Place in the staining solution for 10 minutes.
  5. Rinse well with 95% ethanol.
  6. Counterstain the nuclei and/or the cytoplasm if wished.
  7. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  purple
  • Mast cells  –  purple
  • Pituitary β cells  –  purple
  • Sulphated mucins  –  purple
  • Background  –  as the counterstain
  • Nuclei  –  as the nuclear stain

Notes

  • The basic fuchsin used for this solution should be one that is suitable for Schiff’s reagent, i.e., it should have a high pararosanilin content. Both methods involve forming a compound between an aldehyde and dye.
  • Light counterstaining with a progressive alum hematoxylin and eosin is also suitable.
  • Many other counterstains can be used, including methods such as Masson’s trichrome.
  • Gabe described a technique for the preparation and use of aldehyde fuchsin powder.

Safety Note

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

References

  1. Drury, R.A.B. and Wallington, E.A., (1980)
    Carleton’s histological technique Ed. 5
    Oxford University Press, Oxford, UK.
March 30, 2023
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Mayer’s Alum Hematoxylin Staining Variants

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

The solution usually meant when Mayer’s hemalum is specified is actually a modification of Mayer’s 1901 formula by Langeron. Mayer published several alum hematoxylin variants for nuclear staining, both progressive and regressive. Such solutions usually contain hematoxylin and an alum, and are called hemalum or alum hematoxylin solutions. Many formulae have been suggested. They vary in the amount of hematoxylin, the amount and type of aluminum salts, solvents, oxidizing agents and stabilizers.

Read More
March 30, 2023
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McNulty-Smith’s Zirconyl Hematoxylin

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

McNulty-Smith's Zirconyl Hematoxylin

6
steps
6
materials
print

This hematoxylin substitutes for alcian blue pH 2.5 for staining acid mucins.

Materials

IngredientAmountFunction
Hematoxylin100 mgDye
Ethanol, absolute5 mLSolvent
Sodium iodate, 0.5% aqu.5 mLOxidant
Zirconyl chloride, octahydrate400 mgMordant
Distilled Water22.5 mLSolvent
Glycerol7.5 mLAnti-oxidant

Compounding Procedure

  1. Dissolve the hematoxylin in the ethanol.
  2. Add the sodium iodate solution (freshly made).
  3. Add the zirconyl chloride.
  4. Combine the glycerol and water, and add to the solution.
  5. Stir for 5 minutes.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 10 minutes.
  3. Wash with distilled water, two changes of 2 minutes each.
  4. Counterstain in methylene green for 5 minutes.
  5. Wash with distilled water, two changes of 2 minutes each.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  green
  • Acid mucins  –  Purple

Notes

  • Formalin fixed, paraffin sections are suitable.
  • The counterstain recommended was 0.05% methylene green in 2.4% boric acid.
  • This method was recommended as a substitute for alcian blue staining of acid mucins (pH 2.5).
  • Staining may be removed with 1% hydrochloric acid in 70% ethanol.
  • The solution above is McNulty’s modification of Smith’s original, and stains more intensely:
    • Hematoxylin, 100 mg
    • Absolute ethanol, 5 mL
    • 0.1% aqueous sodium iodate, 5 mL
    • Zirconyl chloride octahydrate, 400 mg
    • 22% aqueous glycerol, 45 mL
    • Glacial acetic acid, 5 mL

Safety Note

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

References

  1. Smith, A. A., (1999)
    Zirconyl hematoxylin staining of acidic mucins.
    Journal of Histochemistry and Cytochemistry, v. 47, p. 1645.
  2. McNulty, J. M. and Smith, A. A., (2004)
    An improved formulation of the zirconyl hematoxylin stain for acidic mucins.
    Biotechnic and Histochemistry, v. 79, Nº 5, p. 191.
  3. McNulty, J. M., Kambour, M. J. and Smith, A. A., (2004)
    Use of an improved zirconyl hematoxylin stain in the diagnosis of Barrett’s esophagus.
    J. Cell. Mol. Med., v. 8, Nº 3, p. 382.
March 30, 2023
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