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Iron Resorcin

French’s Stain for Elastic Fibres

By Dye Type, Elastic Fibers, Iron Resorcin, Protocols, Stain Target, Stain Type
Protocol

French's Stain

for Elastic Fibres

7
steps
9
materials
print

Materials

  • Weigert’s iron hematoxylin or equivalent
  • Van Gieson’s picro-fuchsin
  • French’s solution
    MaterialAmount
    Basic fuchsin1g
    Crystal violet1g
    Resorcin4g
    Ferric chloride, 30% aqu.25mL
    Ethanol 95%200mL
    Distilled water200mL
    Hydrochloric acid, conc.4mL

    Preparation

    1. Add the dye and resorcin to the water in an oversize flask.
    2. Bring to the boil, and add the ferric chloride.
    3. Boil for 5 minutes.
    4. Cool and filter.
    5. Dry the filter paper and beaker. Place the precipitate and filter paper back into the flask.
    6. Add the ethanol and heat carefully until the precipitate dissolves.
    7. Add the hydrochloric acid.
    8. Restore to 200 mL with 95% ethanol.

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 French’s solution until adequately stained (may be overnight).
  3. Wash with 95% ethanol to remove excess solution.
  4. Differentiate with 1% acid alcohol.
  5. Wash in water.
  6. Counterstain with iron hematoxylin and van Gieson.
  7. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  dark blue-green
  • Collagen  –  red
  • Cytoplasm  –  yellow
  • Nuclei  –  blue

Notes

  • This is Weigert’s iron resorcin fuchsin using crystal violet and basic fuchsin instead of basic fuchsin alone.
  • It is usually recommended that the basic fuchsin should not be of the type that produces good Schiff’s reagent. That is, the basic fuchsin should contain more rosanilin than pararosanilin.
  • The ferric chloride solution should be freshly made.

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
March 30, 2023
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Fullmer & Lillie’s Orcinol-New Fuchsin for Elastic Fibres

By Dye Type, Elastic Fibers, Iron Resorcin, Protocols, Stain Target, Stain Type
Protocol

Fullmer & Lillie's Orcinol-New Fuchsin

for Elastic Fibres

5
steps
7
materials
print

Materials

    • Weigert’s iron hematoxylin or equivalent
    • Van Gieson’s picro-fuchsin
    • Orcinol-New Fuchsin solution
      MaterialAmount
      New fuchsin2g
      Orcinol4g
      Ferric chloride, 30% aqu.50mL
      Distilled water200mL
      Ethanol 95%100mL

      Preparation

      1. Add the dye and orcinol to the water in an oversize flask.
      2. Boil for 5 minutes.
      3. Add the ferric chloride.
      4. Boil for a further 5 minutes.
      5. Cool and filter.
      6. Dry the filter paper and beaker. Place the precipitate and filter paper back into the flask.
      7. Add the ethanol and heat carefully until the precipitate dissolves.
      8. Restore to 100 mL with 95% ethanol.

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 orcinol-new fuchsin solution for 15 minutes at 37°C.
  3. Wash with 3 changes of 70% ethanol for 5 minutes each.
  4. Counterstain with iron hematoxylin and van Gieson.
  5. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  deep violet
  • Collagen  –  red
  • Cytoplasm  –  yellow
  • Nuclei  –  black

Notes

  • Fullmer and Lillie specified 25mL USP IX Liquor Ferri Chloridi, or a solution of 15.5g ferric chloride (FeCl3.6H2O) made up to 25 mL with water, i.e. 25mL of a 62% solution. In either case, it should be fresh.

Safety Note

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

References

  1. Fullmer, H M and Lillie, R D, (1956).
    A selective stain for elastic tissue (orcinol-new fuchsin).,
    Stain Technologyl, v 31, page 27-29.
  2. Lillie, R D and Fullmer, H M, (1976).
    Histopathological technic and practical histochemistry, Ed. 4. pp. 714.
    McGraw-Hill, New York, USA.
March 30, 2023
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Hart’s Stain for Elastic Fibres

By Dye Type, Elastic Fibers, Iron Resorcin, Protocols, Stain Target, Stain Type
Protocol

Hart's Stain

for Elastic Fibres

7
steps
4
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 Hart’s solution overnight.
  3. Wash with 95% ethanol to remove excess solution.
  4. Differentiate with 1% acid alcohol.
  5. Wash with tap water.
  6. Counterstain with iron hematoxylin and van Gieson.
  7. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  blue black
  • Collagen  –  red
  • Cytoplasm  –  yellow
  • Nuclei  –  blue

Notes

  • This modification should be used if poor staining is obtained using Weigert’s method.
  • The optimum dilution may be more or less than given.

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., (1974)
    Handbook of histopathological and histochemical techniques Ed. 3
    Butterworth, London, UK.
March 30, 2023
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Humberstone’s Stain for Elastic Fibres

By Dye Type, Elastic Fibers, Iron Resorcin, Protocols, Stain Target, Stain Type
Protocol

Humberstone's Stain

for Elastic Fibres

8
steps
12
materials
print

Materials

  • Weigert’s iron hematoxylin or equivalent
  • Van Gieson’s picro-fuchsin
  • Potassium permanganate, 0.25% aqu., for the Mallory bleach
  • Humberstone’s solution
    MaterialAmount
    Victoria blue 4R1g
    Ethyl violet1g
    Resorcin4g
    Dextrin0.5g
    Ferric chloride, 30% aqu.25mL
    Ethanol, 95%150mL
    Phenol10g
    Hydrochloric acid, conc.4mL
    Distilled water200mL

      • Preparation

      1. In an oversized flask, add the dyes to the water, and boil to dissolve.
      2. Add the resorcin, dextrin and ferric chloride. Boil for 3 minutes. Cool and filter.
      3. Place the precipitate and filter paper back into the original flask. Add 100 ml of 95% ethanol and ently for 15 minutes.
      4. Cool and filter.
      5. Make up the volume to 350 mL with 95% ethanol.
      6. Add the phenol and hydrochloric acid.

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. Do a Mallory bleach with 0.25% potassium permanganate for ten minutes.
  3. Rinse with 95% ethanol.
  4. Place into Humberstone’s solution overnight.
  5. Wash with 95% ethanol to remove excess solution.
  6. Wash in water.
  7. Counterstain with iron hematoxylin and van Gieson.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  blue-black
  • Nuclei  –  black
  • Cytoplasm  –  yellow
  • Collagen  –  red

Notes

  • The authors state that buffered formalin is an unsuitable fixative. I have not found this so and have used this method regularly with such tissue. It may be that the modern practice of minimalist fixation limits the effect.
  • As the solution ages, the demonstration of elastic fibres improves in both quality and speed. When the solution is a few years old elastic is demonstrated within about 4 hours, although overnight is generally
    preferable.
  • The solution is stable for more than ten years. Due to the improvement in staining as the solution ages, it is suggested that a litre, or more, be made at a time.
  • The authors suggest as suitable counterstains, iron hematoxylin and van Gieson, Masson’s trichrome or MSB (with green collagen).

Safety Note

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

References

  1. Humberstone, G. C. W., and Humberstone, F. D., (1969)
    An Elastic Tissue Stain
    Journal of Medical Laboratory Technology, V. 26, No 2, pp. 99.
March 30, 2023
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Miller’s Stain for Elastic Fibres

By Dye Type, Elastic Fibers, Iron Resorcin, Protocols, Stain Target, Stain Type
Protocol

Miller's Stain

for Elastic Fibres

8
steps
12
materials
print

Materials

Preparation

  1. In an oversized flask heat the water and add the three dyes.
  2. When dissolved, add the resorcin, dextrin and ferric chloride in the order given.
  3. Boil for 5 minutes, then filter while hot.
  4. Place the precipitate and filter paper back into the original flask, add the 95% ethanol and simmer gently for 20 minutes.
  5. Cool and filter.
  6. Restore the volume to 200 mL with 95% ethanol, then add 2 mL of concentrated hydrochloric acid.

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. Do a Mallory bleach with 0.25% potassium permanganate for ten minutes.
  3. Rinse with 95% ethanol.
  4. Place into Miller’s solution up to three hours.
  5. Wash with 95% ethanol to remove excess solution.
  6. Wash in water.
  7. Counterstain with iron hematoxylin and van Gieson.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

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

Notes

  • Three hours is recommended, although staining may be adequate after 90 minutes.
  • Dilute with an equal quantity of 95% ethanol for overnight staining.
  • The staining solution is stable for years.

Safety Note

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

References

  1. Miller, P.J., (1971)
    Journal of Medical Laboratory Technology, V. 28
  2. Ellis, R.,
    Miller’s Elastic Staining Protocol
    IHC World
March 30, 2023
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Sheridan’s Stain for Elastic Fibres

By Dye Type, Elastic Fibers, Iron Resorcin, Protocols, Stain Target, Stain Type
Protocol

Sheridan's Stain

for Elastic Fibres

7
steps
9
materials
print

Materials

  • Weigert’s iron hematoxylin or equivalent
  • Van Gieson’s picro-fuchsin
  • Sheridan’s solution
    MaterialAmount
    Crystal violet2g
    Dextrin2g
    Resorcin4g
    Ferric chloride, 30% aqu.25mL
    Ethanol 95%200mL
    Distilled water200mL
    Hydrochloric acid, conc.4mL

Preparation

  1. Add the dye, dextrin and resorcin to the water in an oversize flask. Bring to the boil, and add the ferric chloride.
  2. Boil for 5 minutes.
  3. Cool and filter.
  4. Dry the filter paper and beaker. Place the precipitate and filter paper back into the flask.
  5. Add the ethanol and heat carefully until the precipitate dissolves.
  6. Add the hydrochloric acid.
  7. Restore to 200 mL with 95% ethanol.

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 Sheridan’s solution until adequately stained (may be overnight)).
  3. Wash with 95% ethanol to remove excess solution.
  4. Differentiate with 1% acid alcohol.
  5. Wash in water.
  6. Counterstain with iron hematoxylin and van Gieson.
  7. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  green
  • Collagen  –  red
  • Cytoplasm  –  yellow
  • Nuclei  –  blue

Notes

  • This is similar to Weigert’s iron resorcin fuchsin, but substitutes crystal violet and dextrin for basic fuchsin.
  • The ferric chloride solution should be freshly made.

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
March 30, 2023
Love0

Weigert’s Stain for Elastic Fibres

By Dye Type, Elastic Fibers, Iron Resorcin, Protocols, Stain Target, Stain Type
Protocol

Weigert's Stain

for Elastic Fibres

7
steps
8
materials
print

Materials

  • Weigert’s iron hematoxylin or equivalent
  • Van Gieson’s picro-fuchsin
  • Weigert’s solution
    MaterialAmount
    Basic fuchsin2g
    Resorcin4g
    Ferric chloride, 30% aqu.25mL
    Ethanol 95%200mL
    Distilled water200mL
    Hydrochloric acid, conc.4mL

Preparation

  1. Add the dye and resorcin to the water in an oversize flask. Bring to the boil, and add the ferric chloride.
  2. Boil for 5 minutes.
  3. Cool and filter. Dry the filter paper and beaker. Place the precipitate and filter paper back into the flask.
  4. Add the ethanol and heat carefully until the precipitate dissolves.
  5. Add the hydrochloric acid.
  6. Restore to 200 mL with 95% ethanol.

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 Weigert’s solution for 20 minutes to 1 hour.
  3. Wash with 95% ethanol to remove excess solution.
  4. Differentiate with 1% acid alcohol.
  5. Wash in water.
  6. Counterstain with iron hematoxylin and van Gieson.
  7. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres – blue black
  • Collagen – red
  • Cytoplasm – yellow
  • Nuclei – blue

Notes

  • The procedure often benefits from a Mallory bleach.
  • It is usually recommended that the basic fuchsin should not be of the type that produces good Schiff’s reagent. That is, the basic fuchsin should contain more rosanilin than pararosanilin.
  • The ferric chloride solution should be freshly made.

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., (1974)
    Handbook of histopathological and histochemical techniques, Ed. 3
    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. Humason, G. L., (1967)
    Animal tissue techniques, Ed. 2
    W. H. Freeman and Company, San Francisco, Ca, USA
March 30, 2023
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Dahlia-Iron-Resorcin for Elastic Fibres

By Dye Type, Elastic Fibers, Iron Resorcin, Protocols, Stain Target, Stain Type
Protocol

Dahlia-Iron-Resorcin

for Elastic Fibres

8
steps
9
materials
print

Materials

    • Weigert’s iron hematoxylin or equivalent
    • Van Gieson’s picro-fuchsin
    • Iron-resorcin-dahlia solution
      MaterialAmount
      Dahlia2g
      Resorcin4g
      Ferric nitrate, 30% aqu.50mL
      0.5% aqu. hydrochloric acid200mL
      Methanol180mL
      Acetone20mL
      Hydrochloric acid, conc.4mL

Preparation

  1. Bring 200 mL of 0.5% hydrochloric acid in an oversize flask to the boiling point, remove from the heat and carefully add the dahlia.
  2. Return to the boil, and add the resorcin and ferric nitrate.
  3. Continue boiling for 3 minutes, then cool and filter. Discard the filtrate. Dry the filter paper and beaker, then place the precipitate and filter paper back into the flask.
  4. Add the methanol, acetone and hydrochloric acid.
  5. Heat carefully on a hot plate until the precipitate dissolves.

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. Do a Mallory bleach.
  3. Place into the staining solution for 30 minutes or longer.
  4. Wash with 95% ethanol to remove excess solution.
  5. Differentiate with 1% acid alcohol if necessary.
  6. Wash in water.
  7. Counterstain with iron hematoxylin and van Gieson.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  blue black
  • Collagen  –  red
  • Cytoplasm  –  yellow
  • Nuclei  –  blue

Notes

  • Between the 1st and 2nd editions of their book, Bancroft and Stevens changed the solvent for the precipitate to the following:
    SolventAmount
    2-methoxyethanol100mL
    Distilled water100mL
    Hydrochloric acid, conc.4mL

    Dry the filter paper and beaker, then place the precipitate and filter paper back into the flask. Add the mixture and heat carefully on a hot plate until the precipitate dissolves.

  • 2-methoxyethanol is also known as ethylene glycol monomethyl ether.
  • The authors suggest the following dyes as suitable substitutes for dahlia:
    • Methyl violet
    • Ethyl violet
    • Victoria blue
    • Thionin
  • Note that this method uses ferric nitrate instead of ferric chloride.

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. 1 (1977),
    Churchill Livingstone, London, UK.
  2. Bancroft, J. D. and Stevens, A.,
    Theory and Practice of Histological Techniques, Ed. 2 (1982),
    Churchill Livingstone, London, UK.
March 30, 2023
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