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Metachromasia

Bancroft’s Crystal Violet for Amyloid

By Amyloid, Metachromasia, Protocols, Stain Target, Stain Type
Protocol

Bancroft's Crystal Violet

for Amyloid

7
steps
3
materials
print

Materials

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Cryostat sections usually show brighter metachromasia. Unmounted frozen sections may also be floated in each solution and mounted on a slide just before coverslipping.

Protocol

  1. Bring sections to water via xylene and ethanol, except for cryostat and frozen sections.
  2. Place into crystal violet solution for 5 minutes.
  3. Rinse with distilled water.
  4. Differentiate briefly with dilute acetic acid for 15 – 20 seconds.
  5. Counterstain with methyl green for 5 – 15 minutes.
  6. Wash with distilled water.
  7. Either drain all water from the slide until just damp then blot and mount with Apathy’s or Highman’s medium, or drain all water from the slide until just damp then blot and flood with xylene. Repeat until the section is cleared, then mount with a resinous medium.

Expected Results

  • Amyloid – purple-red
  • Background – green
  • Nuclei – green

Safety Note

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

References

  1. Bancroft, J. D., (1963).
    Stain technology, v. 38, p. 336.London, England.
March 30, 2023
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Bancroft’s Methyl Green for Amyloid

By Amyloid, Metachromasia, Protocols, Stain Target, Stain Type
Protocol

Bancroft's Methyl Green

for Amyloid

6
steps
2
materials
print

Materials

  • Methyl green, 2% aqueous, washed with chloroform to remove crystal violet
  • Acetic acid, 1% aqueous

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Cryostat sections usually show brighter metachromasia. Unmounted frozen sections may also be floated in each solution and mounted on a slide just before coverslipping.

Protocol

  1. Bring sections to water via xylene and ethanol, except for cryostat and frozen sections.
  2. Place into methyl green solution for 1-5 minutes.
  3. If necessary, differentiate in dilute acetic acid until amyloid is red and contrasts well.
  4. Rinse well with tap water.
  5. Drain all water from the slide until just damp and blot dry.
  6. Flood with triethylphosphate, then with xylene, and coverslip using a resinous medium.

Expected Results

  • Amyloid – purple-red
  • Background – green
  • Nuclei – green

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. 222.
    Oxford University Press, London, England.
March 30, 2023
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Highman’s Crystal Violet for Amyloid

By Amyloid, Metachromasia, Protocols, Stain Target, Stain Type
Protocol

Highman's Crystal Violet

for Amyloid

7
steps
5
materials
print

Materials

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Cryostat sections usually show brighter metachromasia. Unmounted frozen sections may also be floated in each solution and mounted on a slide just before coverslipping.

Protocol

  1. Bring sections to water via xylene and ethanol, except for cryostat and frozen sections.
  2. Stain nuclei with Weigert’s iron hematoxylin for 5 minutes.
  3. Wash with water.
  4. Place into crystal violet solution for 1-30 minutes until amyloid is stained.
  5. Rinse well with water.
  6. Drain all water from the slide until just damp and mount with Highman’s medium.
  7. Ring the coverslip to inhibit evaporation of the mounting medium and precipitation of the ingredients.

Expected Results

  • Amyloid – purple-red
  • Background – blue-violet
  • Nuclei – black

Notes

  • Methyl violet may be used instead of crystal violet if preferred.
  • Gray notes that Lieb substituted a solution of 0.3% crystal violet in 0.3% hydrochloric acid for Highman’s crystal violet solution.
  • Highman’s gum syrup is a modification of Apathy’s gum syrup and contains potassium acetate or sodium chloride to stop bleeding of the dye into the mounting medium.

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. 452.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
March 30, 2023
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Lendrum’s’ Methyl Violet for Amyloid

By Amyloid, Metachromasia, Protocols, Stain Target, Stain Type
Protocol

Lendrum's' Methyl Violet

for Amyloid

7
steps
3
materials
print

Materials

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Cryostat sections usually show brighter metachromasia. Unmounted frozen sections may also be floated in each solution and mounted on a slide just before coverslipping.

Protocol

  1. Bring sections to water via xylene and ethanol, except for cryostat and frozen sections.
  2. Place into methyl violet solution for 3 minutes.
  3. Differentiate in formalin until amyloid is red and contrasts well with the tissue.
  4. Place into sodium chloride solution for 5 minutes.
  5. Rinse well with tap water.
  6. Drain all water from the slide until just damp and mount with corn syrup.
  7. Ring the coverslip to inhibit evaporation of the mounting medium and precipitation of the ingredients.

Expected Results

  • Amyloid – purple-red
  • Background – blue-violet
  • Nuclei – blue-violet

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, Allison R T, Barr W T, (1985), p. 466.
    Cellular pathology technique. Ed. 4.,
    Butterworths, London, England.
March 30, 2023
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Birch-Hirschfeld’s Crystal Violet for Amyloid

By Amyloid, Metachromasia, Protocols, Stain Target, Stain Type
Protocol

Birch-Hirschfeld's Crystal Violet

for Amyloid

9
steps
4
materials
print

Materials

Tissue Sample

Frozen sections are preferred. Cryostat sections usually show brighter metachromasia. Unmounted frozen sections may also be floated in each solution and mounted on a slide just before coverslipping. 5µ paraffin sections of neutral buffered formalin fixed tissue are likely also suitable. Other fixatives may be satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol, except for cryostat and frozen sections.
  2. Place into bismarck brown solution for 5 minutes.
  3. Rinse well with 95% ethanol, then rinse with distilled water.
  4. Place into crystal violet solution for 5 minutes.
  5. Rinse with water.
  6. If necessary, differentiate in 1% acetic acid until amyloid is red and contrasts well with the tissue.
  7. Wash well in tap water.
  8. Drain all water from the slide until just damp and mount with levulose syrup.
  9. Ring the coverslip to inhibit evaporation of the mounting medium.

Expected Results

  • Amyloid  –  purple-red
  • Background  –  blue-violet
  • Nuclei  –  brown

Notes

  • Methyl violet may be used instead of crystal violet if preferred.
  • Although levulose syrup (fructose syrup or high fructose corn syrup) is specified it is likely that Highman’s gum syrup would be preferable as it inhibits leaching of the dye.
  • Although bismarck brown is metachromatic (yellow metachromasia), it is used here as a basic dye for staining nuclei.

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.451.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
March 30, 2023
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Jurgens’ Crystal Violet for Amyloid

By Amyloid, Metachromasia, Protocols, Stain Target, Stain Type
Protocol

Jurgens' Crystal Violet

for Amyloid

7
steps
3
materials
print

Materials

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Cryostat sections usually show brighter metachromasia. Unmounted frozen sections may also be floated in each solution and mounted on a slide just before coverslipping.

Protocol

  1. Bring sections to water via xylene and ethanol, except for cryostat and frozen sections.
  2. Place into crystal violet solution for 2-5 minutes.
  3. Rinse well with water and examine microscopically.
  4. If necessary, differentiate in dilute acetic acid until amyloid is red and contrasts well with the tissue.
  5. Wash very well in tap water, about 5 minutes.
  6. Drain all water from the slide until just damp and mount with Highman’s medium.
  7. Ring the coverslip to inhibit evaporation of the mounting medium and precipitation of the ingredients.

Expected Results

  • Amyloid  –  purple-red
  • Background  –  blue-violet
  • Nuclei  –  blue-violet

Notes

  • Methyl violet may be used instead of crystal violet if preferred.
  • Highman’s gum syrup is a modification of Apathy’s gum syrup and contains potassium acetate or sodium chloride to stop bleeding of the dye into the mounting medium.

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. 222.
    Oxford University Press, London, England.
March 30, 2023
Love0