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Direct Dye Staining

Llewellyn’s Sirius Red for Amyloid

By Amyloid, Direct Dye Staining, Eosinophils, Intracytoplasmic Granules, Paneth Cells, Protocols, Stain Target, Stain Type
Protocol

Llewellyn's Sirius Red

for Amyloid

8
steps
3
materials
print

Materials

MaterialAmount
Sirius red F3B0.5g
Distilled water50mL
Ethanol, absolute50mL

Staining Solution Preparation

  1. Dissolve the dye into the water, add ethanol and mix well.
  2. Add 1 mL of 1% sodium hydroxide. Then, while strong backlighting and swirling, add drops of 20% sodium chloride until a fine haze is detected. Usually about 2 mL is adequate. Adding more than 4 mL causes excessive precipitation. The solution is reasonably stable for several months, but slowly deteriorates. Extend the staining time to compensate. When it requires more than 2 hours to adequately stain, prepare a new solution.

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 with a progressive alum hematoxylin for a few minutes.
  3. Rinse with tap water.
  4. Rinse with ethanol.
  5. Place into alkaline sirius red for 1 – 2 hours.
  6. Rinse well with tap water.
  7. Dehydrate with absolute ethanol.
  8. Clear with xylene and mount with a resinous medium.

Expected Results

  • Amyloid  –  red
  • Eosinophil and Paneth cell granules  –  red
  • Nuclei  –  blue
  • Background  –  colorless
   

Notes

  • Amyloid displays deep green birefringence when viewed with crossed polarisers, one above and one below the section.
  • Eosinophils and Paneth cell granules are also demonstrated. If used for this purpose the sodium chloride may be ommitted.
  • This method uses sirius red F3B. The dye Sirius red 4B is not suitable.

Safety Note

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

References

  1. Llewellyn, B.D., (1970)
    An improved sirius red method for amyloid.
    Journal of Medical Laboratory Technology, v 23, 308
March 30, 2023
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Puchtler, Sweat and Levine’s Congo Red for Amyloid

By Amyloid, Direct Dye Staining, Protocols, Stain Target, Stain Type
Protocol

Puchtler, Sweat and Levine's Congo Red

for Amyloid

7
steps
10
materials
print

Materials

  • Mayer’s hemalum
  • Stock alcoholic salt
    • Saturate 80% ethanol with sodium chloride.
  • Stock congo red
    • Saturate 80% ethanol with congo red and sodium chloride.
    • Let stand for 24 hours.
  • Working alkaline alcohol
    MaterialAmount
    Stock alcoholic salt50mL
    1% sodium hydroxide0.5mL

    Use within 15 minutes.

  • Working congo red
    MaterialAmount
    Stock congo red50mL
    1% sodium hydroxide0.5mL

    Use within 15 minutes.

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 with hemalum.
  3. Rinse well with distilled water.
  4. Place in working alkaline alcohol for 20 minutes.
  5. Place in working congo red for 20 minutes.
  6. Dehydrate rapidly with absolute ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Amyloid  –  Deep pink to red
  • Nuclei  –  blue
  • Background  –  colorless

Notes

  • Amyloid displays apple green birefringence when viewed with crossed polarizers, one above and one below the section.
  • This method is considered to be the most reliable of all congo red methods for amyloid.

Safety Note

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

References

  1. Puchtler, H., Sweat, F. and Levine, M., (1962),
    On the binding of congo red by amyloid,
    Journal of Histochemistry and Cytochemistry, v 10, page 355
  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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Stokes’ Congo Red for Amyloid

By Amyloid, Direct Dye Staining, Protocols, Stain Target, Stain Type
Protocol

Stokes' Congo Red

for Amyloid

7
steps
5
materials
print

Materials

  • Mayer’s hemalum
  • 80% ethanol
  • Alkaline Congo Red
    MaterialAmount
    Congo redasrequired
    Ethanol, 80%100mL
    Potassium hydroxide0.2g

Compounding Procedure

  1. Dissolve the potassium hydroxide in the ethanol.
  2. Add sufficient dye to saturate. Leave overnight and filter.
  3. Stable for about 3 months.

Tissue Sample

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

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into alkaline congo red for 25 minutes.
  3. Wash with tap water for 5 minutes.
  4. Stain nuclei with Mayer’s hemalum for 5 minutes.
  5. Blue in running tap water for 10 minutes.
  6. Dehydrate rapidly in absolute ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Amyloid – orange to red
  • Nuclei – blue
  • Background – colorless

Notes

  • If congo red is applied for longer than 25 minutes, the background will show some coloration.
  • Green birefringence is displayed under crossed polarizers.

Safety Note

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

References

  1. Stokes, Gwen, (1976),
    An improved congo red method for amyloid,
    Medical Laboratory Sciences, v 33, 79
March 30, 2023
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Sweat and Puchtler’s Sirius Red for Amyloid

By Amyloid, Direct Dye Staining, Protocols, Stain Target, Stain Type
Protocol

Sweat and Puchtler's Sirius Red

for Amyloid

11
steps
8
materials
print

Materials

  • Mayer’s hemalum
  • Neutral buffered formalin (pH 7.0)
  • 0.1M borate or borate-phosphate buffer pH 9.0
  • Alkaline alcohol
    MaterialAmount
    Ethanol, 80%100mL
    Sodium hydroxide, 1% aqueous1mL
  • Sirius red
    MaterialAmount
    Sirius red1g
    Distilled water100mL
    Sodium chloride0.5g

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 neutral buffered formalin overnight.
  3. Wash in tap water for 15 minutes.
  4. Place into alkaline alcohol for 20 – 60 minutes.
  5. Rinse well with distilled water.
  6. Place into sirius red at 60°C for 60 – 90 minutes.
  7. Rinse with buffer.
  8. Wash with tap water for 5 minutes.
  9. Stain nuclei with hemalum and blue.
  10. Dehydrate with absolute ethanol.
  11. Clear with xylene and mount with a resinous medium.

Expected Results

  • Amyloid  –  red
  • Nuclei  –  blue
  • Background  –  colorless

Notes

  • This method uses sirius red F3B. The dye sirius red 4B is not suitable.
  • Sirius scarlet GG, CI 40270, may also be used.
  • Amyloid displays deep green birefringence when viewed with crossed polarizers, one above and one below the section.

Safety Note

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

References

  1. Sweat, F. and Puchtler, H., (1965),
    Demonstration of amyloid with direct dyes,
    Archives of Pathology, v 80, page 613
March 30, 2023
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Bennhold’s Congo Red for Amyloid

By Amyloid, Direct Dye Staining, Protocols, Stain Target, Stain Type
Protocol

Bennhold's Congo Red

for Amyloid

8
steps
5
materials
print

Materials

  • Mayer’s hemalum
  • Congo red
    MaterialAmount
    Congo red1g
    Distilled water100mL
  • Lithium carbonate
    MaterialAmount
    Lithium carbonateasrequired
    Distilled water100mL

Dissolve to saturation.

Tissue Sample

Paraffin sections of formalin fixed tissues are satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into the congo red solution for
    1. 30 minutes at room temperature, or
    2. 45 minutes at 56°C, or
    3. 15 seconds at boiling.
  3. Drain and, without rinsing, place into lithium carbonate solution for 15 seconds.
  4. Drain and, without rinsing, differentiate in 80% ethanol. This usually takes just a few seconds.
  5. Wash well with tap water.
  6. Stain nuclei with hematoxylin and blue.
  7. Dehydrate with ethanol.
  8. Clear with xylene and mount with a resinous medium.

Expected Results

  • Amyloid  –  red
  • Nuclei  –  blue

Notes

  • Amyloid displays apple green birefringence when viewed with crossed polarisers, one above and one below the section.
  • The congo red is most commonly applied for 30 minutes at room temperature.
  • Differentiation in 80% ethanol is difficult to control, and amyloid is often poorly demonstrated. Due to this the method is not usually recommended.

Safety Note

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

References

  1. Bennhold, H., (1922),
    Eine specifische amyloidfärbung mit kongorot,
    Münchener Medizinische Wochenschrift, v 44, page 1537
March 30, 2023
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Congo Red Fluorescence for Amyloid

By Amyloid, Direct Dye Staining, Protocols, Stain Target, Stain Type

Protocol

Congo Red Fluorescence

for Amyloid

8
steps
4
materials
print

Expected Results

Congo red staining showing amyloid and lipofuscin in an autopsy specimen of senile cardiac amyloidosis

fig1-congo-red-staining-amyloid-lipofuscin-autopsy-senile-cardiac-amyloidosis-600x400

Figure 1. Congo Red Staining Showing Amyloid and Lipofuscin in an Autopsy Specimen of Senile Cardiac Amyloidosis

Congo red staining of an autopsy specimen of senile cardiac amyloidosis, showing amyloid (extracellular washed-out red material) and abundant lipofuscin (yellow granular material). Cardiac amyloidosis very high mag by Nephron / Michael Bonert, Pathology & Molecular Medicine, McMaster University is licensed under CC BY-SA 3.0.

  • Amyloid  –  Orange to red fluorescence

Materials

Congo red

MaterialAmount
Congo red0.1g
Ethanol, 50%100mL

Differentiator

MaterialAmount
Potassium hydroxide0.2g
Ethanol, 80%100mL

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 congo red for 1 minute.
  3. Wash with tap water.
  4. Place into differentiator until the section appears to be unstained.
  5. Wash with water.
  6. Dehydrate with absolute ethanol.
  7. Clear with xylene.
  8. Mount with a fluorescence free resinous medium.

Notes

  • This method is very similar to Highmans procedure.

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. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
March 30, 2023
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Eastwood and Cole Congo Red for Amyloid

By Amyloid, Direct Dye Staining, Protocols, Stain Target, Stain Type
Protocol

Eastwood and Cole Congo Red

for Amyloid

6
steps
7
materials
print

Materials

  • Mayer’s hemalum
  • Buffer pH 10
    MaterialAmount
    Glycine, 0.1M30mL
    Sodium chloride, 0.1M30mL
    Sodium hydroxide, 0.1M40mL
  • Congo red
    MaterialAmount
    Congo red0.5g
    Buffer pH 1050mL
    Ethanol, absolute50mL

    Combine the ethanol and buffer. Dissolve the congo red.

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 hemalum and blue.
  3. Place in congo red solution for 10 – 20 minutes.
  4. Rinse off the staining solution with 70% ethanol until clear.
  5. Dehydrate with ethanol.
  6. Clear with xylene and mount with a resinous medium.

Expected Results

  • Amyloid  –  Orange to red
  • Eosinophils and elastic  –  Orange to red
  • Nuclei  –  Blue

Notes

  • Amyloid displays apple green birefringence when viewed with crossed polarisers, one above and one below the section.

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. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
March 30, 2023
Love0

Highman’s Congo Red for Amyloid

By Amyloid, Direct Dye Staining, Protocols, Stain Target, Stain Type
Protocol

Highman's Congo Red

for Amyloid

7
steps
6
materials
print

Materials

  • Mayer’s hemalum
  • Congo red
    MaterialAmount
    Congo red0.5g
    Distilled water50mL
    Ethanol, 100%50mL
  • Alkaline ethanol
    MaterialAmount
    Ethanol, 80%100mL
    Potassium hydroxide0.2g

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. Place into congo red solution for 5 minutes or longer.
  3. Differentiate in alkaline ethanol (about 5-30 seconds).
  4. Wash well with tap water.
  5. Stain nuclei with hematoxylin and blue.
  6. Dehydrate with ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Amyloid  –  orange red
  • Nuclei  –  blue
  • Background  – colorless

Notes

  • Amyloid displays apple green birefringence when viewed with crossed polarisers, one above and one below the section.
  • Sirius red F3B may also be used in this method.

Safety Note

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

References

  1. Highman, B., (1946),
    Improved methods for demonstrating amyloid in paraffin sections,
    Archives of Pathology, v 41, page 559
  2. Bancroft, J. D. and Stevens, A.
    Theory and practice of histological techniques,
    Churchill Livingstone, London, England
March 30, 2023
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