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Melanin & Enterochromaffin

Masson Fontana Silver Reduction

By Intracytoplasmic Granules, Melanin & Enterochromaffin, Protocols, Stain Target
Protocol

Masson Fontana

Silver Reduction

13
steps
7
materials
print

Materials

  • Silver nitrate, 10% aqu.
  • Strong ammonium hydroxide (s.g. 0.880)
  • Sodium thiosulphate, 3% aqu.
  • Gold chloride, 0.1% aqu.
  • Neutral red, 1% aqu.
  • Mallory bleach
    MaterialAmount
    Potassium permanganate, 1% aqu.47.5mL
    Sulfuric acid, 3% aqueous2.5mL

Preparation of Ammoniacal Silver

  1. Place 20 mL of the 10% silver nitrate in a flask and add drops of strong ammonium hydroxide while swirling the solution.A precipitate will form at first, but will redissolve as more ammonia is added.
  2. Stop when it is almost redissolved and is faintly opalescent, then add 20 mL of distilled water.

Tissue Sample

5µ 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. Optionally, remove melanin with the Mallory bleach.
  3. Bleach in Oxalic acid for a few minutes.
  4. Rinse with distilled water.
  5. Treat with ammoniacal silver, either:
    1. overnight at room temperature, or
    2. 45-60 minutes at 37°C, or
    3. 30 minutes at 56°C
  6. Rinse well with distilled water.
  7. Optionally, tone with 0.1% gold chloride for 10 seconds.
  8. Rinse well with distilled water.
  9. Fix in sodium thiosulfate for 5 minutes.
  10. Wash well with running tap water.
  11. Counterstain with neutral red for 1 minute.
  12. Rinse with tap water.
  13. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Melanin (unbleached) – black
  • Melanin (bleached) – unstained
  • Enterochromaffin – black
  • Lipofuscin – black
  • Nuclei – red
  • Background – grey

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. (1977).
    Theory and practice of histological techniques.
    Churchill Livingstone, Edinburgh, UK.
  2. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide. p. 596
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
March 30, 2023
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Methenamine Silver for Reducing Substances

By Intracytoplasmic Granules, Melanin & Enterochromaffin, Protocols, Stain Target
Protocol

Methenamine Silver

for Reducing Substances

13
steps
7
materials
print

Materials

  • Yellow gold chloride, 0.2% aqu.
  • Sodium thiosulfate, 3% aqu.
  • Stock Methenamine silver
    MaterialAmount
    Methenamine, 3% aqu.100mL
    Silver nitrate, 5% aqu.5mL

    Shake until the precipitate redissolves. Silvering of the container indicates deterioration.

  • Working Methenamine silver
    MaterialAmount
    Stock Methenamine silver50mL
    Borax, 5% aqu.5mL

    Make just before use and preheat to 56°C.

  • Mallory bleach
    MaterialAmount
    Potassium permanganate, 1% aqu.47.5mL
    Sulfuric acid, 3% aqu.2.5mL

Tissue Sample

5µ 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. Optionally, remove melanin with the Mallory bleach.
  3. Bleach in Oxalic acid for a few minutes.
  4. Rinse with distilled water.
  5. Treat with methenamine silver solution at 56&degC. until impregnated
  6. Wash with distilled water.
  7. Tone in gold chloride solution for 1 minute.
  8. Rinse with distilled water.
  9. Fix in sodium thiosulfate for 5 minutes.
  10. Wash well with running tap water.
  11. Counterstain with neutral red for 1 minute
  12. Rinse with tap water.
  13. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Melanin (unbleached) – Black
  • Melanin (bleached) – Unstained
  • Enterochromaffin – Black
  • Lipofuscin – Black
  • Nuclei – Red

Safety Note

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

References

  1. Histology Bench Manual.
    Prince George Regional Hospital
March 30, 2023
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Diazonium Reaction for Enterochromaffin

By Intracytoplasmic Granules, Melanin & Enterochromaffin, Protocols, Stain Target
Protocol

Diazonium Reaction

for Enterochromaffin

7
steps
3
materials
print

Materials

  • Mayer’s hemalum or similar
  • Solution A
    MaterialAmount
    Fast red B, 1% aqu.5mL
    Lithium carbonate, sat. aqu.2mL

    Refrigerate the stock solutions at 4°C. Use the working solution immediately it is prepared.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable.


Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Rinse with distilled water.
  3. Treat with solution A for 1 minute at 4°C.
  4. Rinse well with distilled water.
  5. Stain with Mayer’s hemalum for 1 minute.
  6. Wash well with running tap water.
  7. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Enterochromaffin – Orange-red
  • Nuclei – Blue

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. (1977).
    Theory and practice of histological techniques.
    Churchill Livingstone, Edinburgh, UK.
March 30, 2023
Love0

Schmorl’s Stain for Reducing Substances

By Intracytoplasmic Granules, Melanin & Enterochromaffin, Metal Impregnation, Metal Impregnation, Non-Silver, Protocols, Stain Target, Stain Type
Protocol

Schmorl's Stain

for Reducing Substances

6
steps
4
materials
print

Materials

  • Nuclear fast red
  • Schmorl’s solution
    MaterialAmount
    Ferric chloride, 1% aqueous, fresh30mL
    Potassium ferricyanide, 1% aqueous, fresh4mL
    Distilled water6mL

This solution should be freshly prepared immediately before use.

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 distilled water via xylene and ethanol.
  2. Place into freshly made Schmorl’s solution for 10 minutes.
  3. Wash well with water.
  4. Counterstain with nuclear fast red.
  5. Rinse well with water.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Reducing substances  –  blue
  • Nuclei  –  red

Notes

  • Reducing substances which may be present are colored blue. This includes melanin, for which it is a useful method, enterochromaffin and lipofuscin.
  • The Schmorl’s solution is from Lillie’s modification of Schmorl’s ferricyanide reduction method for tissue reducing substances.

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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