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Stain Target

Eastwood and Cole Congo Red for Amyloid

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

Eastwood and Cole Congo Red

for Amyloid

6
steps
7
materials

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.

Highman’s Congo Red for Amyloid

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

Highman's Congo Red

for Amyloid

7
steps
6
materials

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

Hughesdon’s Metachromatic Stain

By Intracytoplasmic Granules, Mast Cells, Protocols, Stain Target

Hughesdon's Metachromatic Stain

8
steps
4
materials

Materials

Staining solution

MaterialAmount
Azure A0.2g
Distilled water100mL

Differentiating solution

MaterialAmount
Uranyl nitrate0.2g
Distilled water100mL

Tissue Sample

Paraffin sections of neutral buffered formalin fixed tissue are suitable. Mercuric chloride fixatives are reputed to emphasise metachromasia. Other fixatives may be satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Do a Mallory bleach using 1% potassium permangate alone for 5 minutes.
  3. Place in to staining solution for 5 minutes.
  4. Rinse with tap water.
  5. Place into the differentiating solution for 10 seconds.
  6. Rinse with tap water.
  7. Blot dry, dehydrate with absolute ethanol.
  8. Clear with xylene and mount with a synthetic resinous medium.

Expected Results

  • Acid mucopolysaccharides – Red/purple
  • Mast cells – Red/purple
  • Background – Blue

Notes

  • The time of differentiation should be adjusted to give good color contrast.
  • Metachromatically stained materials include acid mucins, cartilage, connective tissue ground substance, and mast cell granules.
  • It may be difficult to obtain uranyl nitrate due to restrictions placed on its distribution in some jurisdictions.

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. 5. pp. 2250
    Oxford University Press, London, England.

Fluorescent Schiff

By Aldehydes, Chromic Acid-Schiff Reaction, Protocols, Schiff's Reagent Reactions, Stain Target, Stain Type

Fluorescent Schiff

11
steps
7
materials

This method demonstrates fungi, and may also be called a Fluorescent Gridley or CAFS.

Materials

  • Fluorescent Schiff reagent
  • Solution A
    MaterialAmount
    Chromium trioxide50g
    Distilled water500mL
  • Solution B
    MaterialAmount
    Sodium sulfite5g
    Distilled water500mL
  • Solution C
    MaterialAmount
    Ethanol, 70%495mL
    Hydrochloric acid5mL

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 in solution A for 10 minutes.
  3. Wash well with tap water.
  4. Bleach with solution B for 1 minute.
  5. Wash well with tap water.
  6. Rinse with distilled water.
  7. Place in fluorescent Schiff reagent for 20 minutes.
  8. Wash well with tap water.
  9. Place in solution C, two changes, 5 minutes each.
  10. Wash well with tap water.
  11. Dehydrate, clear and mount in a non fluorescent resinous medium.

Expected Results

Using a BG 12 exciter filter, and OG 4 (yellow) and/or OG5 (orange) barrier filter, fungi fluoresce yellow with acriflavine and green-red with acridine orange.

Notes

  • This is most useful as a screening method.
  • If background fluorescence is too bright for fungi to be distinguished,it may be quenched with an alum hematoxylin for 1 minute,or 0.5% aqueous potassium permanganate for 1 minute.Quench immediately before the final dehydration step.This should be done with caution as it may reduce fungal fluorescence.

Safety Note

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

References

No specific reference is given for this method. A similar technique using periodic acid as the oxidant is found in:

  1. Culling C.F.A., (1974)
    Handbook of histopathological and histochemical techniques Ed. 3
    Butterworth, London, UK.

Masson 44/41 for Fibrin

By Fibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Masson 44/41

for Fibrin

12
steps
12
materials

With this trichrome stain full and complete fixation is absolutely essential. Minimalist formalin fixation and quick processing will give poorly stained erythrocytes with red or orange tinges instead of the yellow they should have. Lendrum and coworkers specified about seven days fixation in formal sublimate followed by processing thoroughly, sectioning, degreasing with trichlorethylene for 48 hours, then refixing in picro-mercuric-alcohol. Results are usually poor with formalin fixed material, even if treated with Bouin’s fluid at 56°C for an hour or so.

Materials

  • An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
  • Picro-mercuric ethanol
    MaterialAmount
    Ethanol, absolute100mL
    Picric acidto saturation
    Mercuric chlorideto saturation
  • Plasma stain
    MaterialAmount
    Ponceau 6R1g
    Acetic acid, glacial1mL
    Distilled water99mL
  • Fibre stain
    MaterialAmount
    Naphthalene blue black CS1g
    Acetic acid, glacial1mL
    Distilled water99mL
  • Polyacid
    MaterialAmount
    Phosphotungstic acid1g
    Distilled water100mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate for one week. They should be paraffin processed with a schedule that thoroughly and completely dehydrates, then thoroughly cleared with xylene and infiltrated with paraffin wax. This process would normally take 48 hours or longer. Err on the side of completeness, and do not attempt to shorten the process. Avoid rapid fixation and overnight processing, as this produces tissues that stain poorly. Sections should be 3-5 µ thick.

Protocol

  1. Dewax sections with xylene.
  2. Place into a sealed container of trichlorethylene at 56°C for 48 hours.
  3. Rinse well with absolute ethanol.
  4. Refix sections in picro-mercuric-ethanol for a minimum of 3 and preferably 24 hours.
  5. Remove mercury pigment with the iodine-thiosulphate sequence.
  6. Stain nuclei with an acid resistant nuclear stain.
  7. Place in the plasma stain for 5 minutes.
  8. Differentiate with the polyacid for 5 minutes.
  9. Place in the fibre stain for 30 minutes.
  10. Rinse briefly with 1% aqueous acetic acid.
  11. Dehydrate with ethanol.
  12. Clear with xylene and mount with a resinous medium.

Expected Results

  • Fresh fibrin  –  red
  • Older fibrin  –  black
  • Connective tissue  –  pale blue
  • Plasma cell inclusions  –  red

Notes

  • Ponceau 6R is also known as acid red 44.
  • Naphthalene blue black CS is also known as acid black 41.

Safety Note

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

References

  1. Lendrum, A. C., et. al. (1962)
    Studies on the character and staining of fibrin.
    Journal of clinical pathology, v. 15, p. 401.

Periodic Acid Fluorescent Schiff

By Aldehydes, Periodic Acid-Schiff Reaction, Protocols, Schiff's Reagent Reactions, Stain Target, Stain Type

Periodic Acid Fluorescent Schiff

9
steps
2
materials

Materials

Tissue Sample

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

Protocol

  1. Dewax and hydrate sections.
  2. Place in periodic acid for 10 minutes.
  3. Wash well with tap water.
  4. Rinse with distilled water.
  5. Place in fluorescent Schiff reagent for 20 minutes.
  6. Wash well with tap water.
  7. Place in acid alcohol, two changes, 5 minutes each.
  8. Wash well with tap water.
  9. Dehydrate, clear and mount in a non fluorescent resinous medium.

Expected Results

Using a BG 12 exciter filter, and OG 4 (yellow) and/or OG5 (orange) barrier filter, 1-2-glycols fluoresce yellow with acriflavine and green-red with acridine orange.

Periodic acid fluorescent schiff

Notes

  • The same materials fluoresce as would be red or pink in a regular PAS reaction.
  • If background fluorescence is too bright it may be quenched with alum hematoxylin for 1 minute,or 0.5% aqueous potassium permanganate for 1 minute. Quench immediately before the final dehydration step.This should be done with caution as it may reduce fluorescence.

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., (1974)
    Handbook of histopathological and histochemical techniques Ed. 3
    Butterworth, London, UK.