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Plasma Cells

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
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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
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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
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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
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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
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Hitchcock Ehrich for Plasma Cells

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

Hitchcock Ehrich

for Plasma Cells

5

steps
3

materials
print

This method uses malachite green and acridine red to stain plasma cells in a similar manner to the methyl green pyronin methods.


Materials

Solution A

MaterialAmount
Malachite green1g
Distilled water100mL

Solution B

MaterialAmount
Acridine red3g
Distilled water100mL

Combine 1 part of solution A with 3 parts of solution B immediately before use.

Tissue Sample

Zenker fixation is recommended. Other fixatives may not be satisfactory.


Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into the staining solution for 30 seconds.
  3. Rinse with water.
  4. Dehydrate rapidly with absolute ethanol.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  green
  • Plasma cell cytoplasm  –  crimson
  • 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. Biological Staining Methods, 6th ed. (1957)
    Gurr George T.,
    George T. Gurr, London, UK
March 30, 2023
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Kurnick Stain for Plasma Cells

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

Kurnick Stain

for Plasma Cells

5
steps
3
materials
print

Materials

Stock pyronin

MaterialAmount
Pyronin Y2g
Distilled water100mL

Stock methyl green

MaterialAmount
Methyl green2g
Distilled water100mL

Working solution

MaterialAmount
Stock pyronin12.5mL
Stock methyl green7.5mL
Distilled water30mL

Storing stock solutions

The pyronin stock solution should be extracted with chloroform ten times to ensure purity. The methyl green stock solution should be extracted a minimum of six times, until the chloroform is no longer colored with any crystal violet. Store both solutions over a layer of chloroform.

Tissue Sample

Most fixatives should be satisfactory if fixation time is not extended, but avoid potassium dichromate or picric acid. Overnight with a 10% formalin variant should be satisfactory.

Protocol

  1. Bring sections to distilled water via xylene and ethanol.
  2. Place into the staining solution for 6 minutes in a coplin jar.
  3. Blot gently.
  4. Dehydrate with n-butanol, two changes of 5 minutes each.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei – green to blue-green
  • Plasma cell cytoplasm, Nissl substance, nucleoli – red
  • Other cell cytoplasm – pink to unstained

Notes

  • As with other methyl green-pyronin methods used for demonstrating nucleic acids, in critical applications a control section should be treated with a 0.1% solution of ribonuclease for one hour at 37°C to remove RNA, and another treated with distilled water alone. Both control sections should be compared with an untreated but stained section.

Safety Note

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

References

  1. Disbrey, B. D., (1970)
    Histological laboratory methods. pp.187-188.
    E. & S. Livingstone, Edinburgh and London, UK.
March 30, 2023
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Trevan and Sharrock’s Methyl Green – Pyronin

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

Trevan and Sharrock's

Methyl Green – Pyronin

6
steps
8
materials
print

Materials

Methyl green stock

MaterialAmount
Methyl green2g
Distilled water100mL

Purifying the methyl green

Before making the methyl green-pyronin stock solution the methyl green must be purified to remove any methyl violet which has formed.

  1. Place the 2% aqueous stock methyl green solution in a stoppered separating funnel.
  2. Add some chloroform and shake periodically for about a half hour.
  3. Crystal violet will discolor the chloroform as it is removed.
  4. Allow to separate, then drain off the methyl green and discard the chloroform.
  5. Repeat the extraction until the chloroform is colorless or almost so.

Pyronin Y stock

MaterialAmount
Pyronin Y5g
Distilled water100mL

Acetate buffer pH4.8

MaterialAmount
0.1M sodium acetate119mL
0.1M acetic acid81mL

Methyl green–pyronin stock

MaterialAmount
Methyl green, 2% aqueous, purified10mL
Pyronin Y, 5% aqueous17.5mL
Distilled water250mL

Staining solution

MaterialAmount
Methyl green–pyronin stock1volume
Acetate buffer pH 4.81volume

Make fresh.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Avoid mercuric chloride.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Rinse with distilled water, then gently blot dry.
  3. Place into the staining solution for 20–30 minutes,
  4. Rinse with distilled water, then gently blot dry.
  5. Dehydrate rapidly with acetone.
  6. Clear with xylene and mount with a resinous medium.

Expected Results

  • DNA  –  blue–green
  • RNA  –  Red

Notes

  • Mercuric chloride fixation may cause the DNA to depolymerize during fixation and should be avoided. The differential staining depends on differences in the degree of polymerization of the two nucleic acids and depolymerization may cause the DNA to stain with pyronin.
  • Acid decalcified tissues are often unsatisfactory and may stain excessively red with pyronin. EDTA decalcification is usually satisfactory.
  • The extracted solutions of methyl green are reasonably stable, but changes in the expected nuclear coloration may require a freshly extracted solution of methyl green to be prepared.
  • This method is reasonably selective for nucleic acids but should be controlled with ribonuclease and/or deoxyribonuclease enzyme extractions if specificity is required.
  • Since this technique is quite simple, it is often recommended for plasma cells. Nissl substance also stains well.

Safety Note

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

References

  1. Drury, R A, and Wallington, E A, (1967).
    Carleton’s histological technique., Ed. 4, p. 164.
    Oxford University Press, London, England.
March 30, 2023
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Einarson’s Gallocyanin-Chrome Alum

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

Einarson’s

Gallocyanin-Chrome Alum

5
steps
3
materials
print

Materials

Solution

MaterialAmount
Chrome alum5g
Gallocyanin0.15g
Distilled water100mL

Solution Preparation

  1. Add the chrome alum and gallocyanin to the water.
  2. Bring to a boil and simmer for 20 minutes.
  3. Cool and filter.

Tissue Sample

5µ paraffin sections of neutral buffered formalin or Zenker 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 staining solution for 24-48 hours.
  3. Rinse well with water.
  4. Dehydrate with ethanols.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nucleic acids  –  blue

Notes

  • Chrome alum is chromium potassium sulfate dodecahydrate, CrK(SO4)2·12H2O
  • Culling states that the pH is 1.64, and that changing this will eliminate or accentuate non-specific staining. Addition of up to 10 mL N hydrochloric acid will eliminate background staining, and addition of up to 5 mL N sodium hydroxide will accentuate it. In either case, staining of nucleic acids is not affected.

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:
    Einarson, (1932)
    American Journal of Pathology, v. 8, p. 295
    Boston, USA
  2. Culling C.F.A., (1974)
    Handbook of histopathological and histochemical techniques Ed. 3
    Butterworth, London, UK.
March 30, 2023
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Roque’s Stain for Cell Inclusions

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

Roque's Stain

for Cell Inclusions

7
steps
6
materials
print

Materials

Stock solution A

MaterialAmount
Citrate buffer, pH 5.8100mL
Methyl green, purified0.1g
Thionin0.0165g

Dissolve the thionin in a small amount of water. Add the buffer and methyl green. Shake and filter. Use fresh.

Citrate buffer, pH 5.8

MaterialAmount
Hydrochloric acid, 0.01M42mL
Sodium citrate, 0.01M58mL

Dehydrant

MaterialAmount
Tertiary butanol80mL
Ethanol, absolute20mL

Tissue Sample

Fix 2mm thick pieces of tissue in 10% formalin containing 1% sodium acetate for 3 hours. Fix smears with methanol.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into the staining solution for 30 minutes at 40°C.
  3. Rinse briefly with distilled water.
  4. Place into dehydrant for 30 seconds
  5. Replace dehydrant, 2 changes, 3 minutes each.
  6. Rinse with absolute ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue-green
  • Nucleolar and cytoplasmic basophil substances  –  red-purple

Notes

  • The reference specifies methyl green, but gives the CI number for ethyl green.
  • The methyl green should be purified, but as a powder rather than as a solution:
    • Add about 10g methyl green to 200 mL chloroform in an Erlenmeyer flask.
    • Shake well.
    • Filter under vacuum and in a fume chamber.
    • Repeat until the chloroform is blue-green instead of violet.

Safety Note

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

References

  1. Humason, G. L., (1967).
    Animal Tissue Techniques., pp. 278
    W. H. Freeman and Company, San Francisco, CA, USA.
March 30, 2023
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