Skip to main content
search
Category

Intracytoplasmic Granules

Langeron’s Stain for Cell Inclusions

By Intracytoplasmic Granules, Plasma Cells, Protocols, Stain Target
Protocol

Langeron's Stain

for Cell Inclusions

6
steps
7
materials
print

Materials

Stock solution A

MaterialAmount
Distilled water100mL
Methyl green4g
Phenol5g

Stock solution B

MaterialAmount
Distilled water100mL
Pyronin Y4g
Phenol5g

Working solution

MaterialAmount
Stock solution A25mL
Stock solution B25mL

Differentiator

MaterialAmount
Ethanol, absolute25mL
Acetone25mL

Tissue Sample

Most fixatives should be satisfactory.


Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into the staining solution for 15 minutes at 50°C.
  3. Rinse briefly with distilled water.
  4. Differentiate until staining is clear.
  5. Dehydrate with amyl alcohol.
  6. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei – violet
  • Plasma cell cytoplasm – red
  • Other cell cytoplasm – pink

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. p. 355
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
March 30, 2023
Love0

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

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
Love0

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
Love0

Eosin for Eosinophils

By Eosinophils, Intracytoplasmic Granules, Protocols, Stain Target
Protocol

Eosin

for Eosinophils

6
steps
2
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. Stain nuclei lightly with alum hematoxylin.
  3. Differentiate if necessary and blue.
  4. Place in eosin solution for 5 minutes.
  5. Wash well with tap water until sections are almost unstained.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei – blue
  • Eosinophils – bright pink
  • Background – pale pink to colorless

Notes

  • The tap water should be hard and very slightly alkaline.
  • Areas which have acidic or soft tap water should use Scott’s tap water substitute. If this is too alkaline, it may be diluted.
  • The water wash is to remove eosin from connective tissues so that the eosinophil granules are the most prominent feature. A light pink background helps with orientation.
  • Adjust staining and washing times to obtain suitable results.

Safety Note

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

March 30, 2023
Love0

Maximow’s Metachromatic Stain

By Intracytoplasmic Granules, Mast Cells, Protocols, Stain Target
Protocol

Maximow's Metachromatic Stain

6
steps
2
materials
print

Materials

  • Ethanol, 50%, saturated with thionin.
  • Sodium carbonate, 0.3% w/v aqueous.

Tissue Sample

Alcohol fixed tissues are recommended, but5µ paraffin sections of neutral buffered formalin fixed tissue may be suitable. Other fixatives may be satisfactory.

Protocol

  1. Dewax with xylene and bring sections to 70% ethanol.
  2. Stain with the thionin solution for 24 to 48 hours.
  3. Blot section dry.
  4. Rinse twice with 95% ethanol.
  5. Rinse well with absolute ethanol.
  6. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei – blue
  • Mast cell granules – red/purple

Safety Note

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

References

  1. McManus, J.F.A. and Mowry, R.W., (1960),
    Staining methods, histologic and histochemical, pp. 261.
    Harper & Row, New York, NY, USA.
March 30, 2023
Love0

Phenolic MGP Solutions

By Intracytoplasmic Granules, Plasma Cells, Protocols, Stain Target
Protocol

Phenolic MGP Solutions

7
steps
7
materials
print

Materials

Table 1. Comparison of phenolic methyl green –pyronin solutions for plasma cells

MaterialFormula
PappenheimUnnaSandifordScott & FrenchLangeronKurnick
Distilled water100mL82mL75mL80mL100mL100mL
Glycerol–mL16mL20mL16mL–mL–mL
Ethanol, 100%0mL2mL0mL4mL–mL–mL
Ethanol, 95%–mL–mL5mL–mL–mL–mL
Phenol0.25g0.4g1.5g1.6g5g–g
Methyl green0.3g0.12g0.15g0.8g2g0.6g
Pyronin Y0.7g0.2g0.5g0.2g2g1g

Tissue Sample

Most fixatives should be satisfactory.

Protocol

These solutions have been included for comparison purposes, and the formulae have been adjusted to make 100 mL of each solution to facilitate the comparison. See the individual methods for details of their use and the actual formula. There is no single method for their application, but the details below may be used as a starting point.

  1. Bring sections to water via xylene and ethanol.
  2. Place in the MGP solution for 10 minutes at room temperature.
  3. Rinse with distilled water.
  4. Differentiate with absolute ethanol if required.
  5. Complete dehydration with acetone if necessary.
  6. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue-green
  • Plasma cell cytoplasm  –  red
  • Other cell cytoplasm  –  pink

Notes

  • The time and temperature in step 2 may need to be increased.
  • Differentiation may not be required, depending on the time and temperature of staining.
  • Various fluids have been used for dehydration: acetone, n-butanol, t-butanol, 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. p. 355
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
  2. Disbrey, B. D., (1970)
    Histological laboratory methods.
    E. & S. Livingstone, Edinburgh and London, UK.
March 30, 2023
Love0

Unna’s Stain for Plasma Cells

By Intracytoplasmic Granules, Plasma Cells, Protocols, Stain Target
Protocol

Unna's Stain

for Plasma Cells

5
steps
6
materials
print

Materials

Staining Solution

MaterialAmount
Distilled water100mL
Phenol0.5g
Glycerol20mL
Ethanol, absolute2.5mL
Pyronin Y0.25g
Methyl green0.15g

Preparation

  1. Place both dyes in a mortar, add the ethanol and grind together.
  2. Heat the glycerol to 50°C and add small volumes to the dyes while grinding.
  3. Dissolve the phenol in the water and wash the dyes from the mortar. Filter.

Tissue Sample

Most fixatives should be satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into the staining solution at 30°C for 10 minutes.
  3. Rinse briefly with water.
  4. Place in absolute ethanol until differentiated.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  violet
  • Plasma cell cytoplasm  –  red
  • Other cell cytoplasm  –  pink

Notes

  • In their description of this method, Carleton and Leach omit the method of preparation, implying that the trituration of the dyes may not be necessary.

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. p. 355
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
  2. Carleton, H M, and Leach, E H, (1938).
    Histological technique., Ed. 2.
    Oxford University Press, London, England.
March 30, 2023
Love0

Sandifords Stain for Plasma Cells

By Intracytoplasmic Granules, Plasma Cells, Protocols, Stain Target
Protocol

Sandifords Stain

for Plasma Cells

5
steps
6
materials
print

Materials

Staining solution

MaterialAmount
Distilled water75mL
Glycerol20mL
Ethanol, 95%5mL
Methyl green0.15g
Pyronin Y0.5g
Phenol1.5g

Tissue Sample

Most fixatives should be satisfactory if fixation is not extended. Reagents which cause depolymerisation of DNA should be avoided. Decalcification may interfere with staining.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into the staining solution for 5-10 minutes.
  3. Rinse briefly with water.
  4. Dehydrate rapidly with acetone.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  violet
  • Plasma cell cytoplasm  –  red
  • Other cell cytoplasm  –  pink

Notes

  • The reference gave no details of the method to use. The details above are from that given for Pappenheim’s solution, and should be suitable as a starting point.
  • Time and temperature of staining may need to be increased.

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. p. 355
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
March 30, 2023
Love0

Pappenheim Stain for Plasma Cells

By Intracytoplasmic Granules, Plasma Cells, Protocols, Stain Target
Protocol

Pappenheim Stain

for Plasma Cells

5
steps
4
materials
print

Materials

Stock solution A

MaterialAmount
Methyl green1g
Distilled water100mL
Phenol0.25g

Stock solution B

MaterialAmount
Pyronin Y4g
Distilled water100mL
Phenol5g

Working solution

MaterialAmount
Stock solution A15mL
Stock solution B35mL

Tissue Sample

Most fixatives should be satisfactory.


Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into the staining solution for 5-10 minutes.
  3. Rinse briefly with water.
  4. Dehydrate rapidly with acetone.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  violet
  • Plasma cell cytoplasm  –  red
  • Other cell cytoplasm  –  pink

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. p. 355
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
March 30, 2023
Love0