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Amyloid

Lynch & Inwood’s Gold for Amyloid

By Amyloid, Metal Impregnation, Metal Impregnation, Non-Silver, Protocols, Stain Target, Stain Type
Protocol

Lynch & Inwood's Gold

for Amyloid

7
steps
5
materials
print

Materials

  • Chloro-auric acid, 1% aqueous
  • Hydrogen peroxide, 3% aqueous
  • Iodine solution
    MaterialAmount
    Iodine1g
    Potassium iodide2g
    Distilled water100mL

    Mix the potassium iodide and iodine crystals together. Add 5 mL of the water and mix until both have dissolved. Add the rest of the water.

Tissue Sample

5-10µ sections from formalin fixed, paraffin embedded tissues, mounted on slides are suitable.


Protocol

  1. Bring sections to water through xylene and ethanols.
  2. Place in the iodine solution for 2½-5 minutes.
  3. Rinse with distilled water, 3 times of 5-10 seconds each.
  4. Place in chloro-auric acid solution for 2½-5 minutes.
  5. Rinse with distilled water, 3 times of 5-10 seconds each.
  6. Place in fresh 3% hydrogen peroxide at 37°C for 6-36 hours.
  7. Dehydrate with ethanol, clear with xylene and mount using a resinous medium.

Expected Results

  • Amyloid – golden yellow to faint purple.
  • Erythrocytes, muscle and liver – blue
  • Nuclei and cytoplasm – brown
  • Collagen – grey

Safety Note

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


References

  1. Lynch, M.J. and Inwood, M.J.H., (1963),
    Gold as a permanent stain for amyloid,
    Stain Technology, v 38, page 260.
March 30, 2023
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King’s Silver Impregnation for Amyloid

By Amyloid, Metal Impregnation, Metal Impregnation, Silver, Protocols, Stain Target, Stain Type
Protocol

King's Silver Impregnation

for Amyloid

7
steps
10
materials
print

Materials

  • Pyridine
  • Ethanol 50%, 75%, 95%
  • Sodium carbonate
    MaterialAmount
    Sodium carbonate, anhydrous3.5g
    Distilled water100mL
  • Silver nitrate, 10%
    MaterialAmount
    Silver nitrate10g
    Distilled water100mL
  • Carbol xylene
    MaterialAmount
    Xylene3volumes
    Phenol1volume
  • Ammoniacal Silver
    MaterialAmount
    Silver nitrate, 10% aqueous5mL
    Strong ammonia (S.G. 0.880)asrequired
    Sodium carbonate, aqueous6.8mL
    Distilled waterasrequired

    Add ammonium hydroxide drop by drop to 5 mL of the silver solution until the precipitate which forms just redissolves. Add 6.8 mL sodium carbonate solution and sufficient water to make up to 75 mL. For use, add a few drops of pyridine to each 10 mL of the solution.

Tissue Sample

10-15µ free floating frozen sections of formalin fixed tissues. Collect into water and wash in a few changes of distilled water to remove residual formalin.


Protocol

  1. Add pyridine to 10 mL ammoniacal silver solution and warm to 40°C.
  2. Float sections in the warm ammoniacal silver until they turn brown.
  3. Rinse with water.
  4. Place in 50% ethanol for 5-10 minutes.
  5. Quickly treat with 75% and 95% ethanols.
  6. Clear with carbol xylene.
  7. Mount sections on to slides and coverslip, using a resinous medium.

Expected Results

  • Amyloid – brown to black.

Safety Note

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


References

  1. King, L.S., (1948),
    Atypical Amyloid Disease: With Observation on a New Silver Stain for Amyloid, American Journal of Pathology, v 24, page 1095-1115
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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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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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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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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Burns, Pennock & Stoward’s Thioflavine T for Amyloid Fluorescence

By Amyloid, Fluorescent Staining, Protocols, Stain Target, Stain Type
Protocol

Burns, Pennock & Stoward's Thioflavine T

for Amyloid Fluorescence

6
steps
3
materials
print

Materials

Tissue Sample

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


Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with alum hematoxylin.
  3. Rinse well with water.
  4. Stain with thioflavine T solution for 5 minutes.
  5. Blot, rinse well with absolute ethanol.
  6. Mount in a fluorescence free resinous mounting medium.

Expected Results

Using a UG1 or UG2 exciter filter and a UV barrier filter, or a BG12 exciter and an OG4 or OG5 barrier filter, amyloid fluoresces lime green or blue.

Safety Note

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


References

  1. Burns, J., Pennock, C.A. and Stoward, P.J., (1967),
    The specificity of the staining of amyloid deposits with thioflavine T,
    Journal of pathology and bacteriology, v 94, page 337.
  2. Bancroft, J.D. and Stevens A. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
March 30, 2023
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Vassar & Culling’s Thioflavine T for Amyloid Fluorescence

By Amyloid, Fluorescent Staining, Protocols, Stain Target, Stain Type
Protocol

Vassar & Culling's Thioflavine T

for Amyloid Fluorescence

8
steps
3
materials
print

Materials

Tissue Sample

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

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into alum hematoxylin for 2 minutes.
  3. Rinse well with water.
  4. Place into thioflavine T solution for 3 minutes.
  5. Rinse with water.
  6. Place into acetic acid solution for 20 minutes.
  7. Wash with water.
  8. Mount in a fluorescence free aqueous mounting medium.

Expected Results

Using a UG1 or UG2 exciter filter and a UV barrier filter, or a BG12 exciter and an OG4 or OG5 barrier filter, amyloid fluoresces bright yellow.

Notes

  • The pretreatment with alum hematoxylin suppresses nuclear fluorescence.
  • Some workers have reported that materials other than amyloid may fluoresce yellow. The authors say this is caused by using a yellow barrier filter and strongly recommended the first filter combination. With this, these materials fluoresce white or pale blue.

Safety Note

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

References

  1. Vassar, P.S. and Culling, C.F.A., (1959),
    Fluorescent stains with special reference to amyloid and connective tissue,
    Archives of pathology, v 68, page 487
  2. Bancroft, J.D. and Stevens A. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
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

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
Love0