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Stain Type

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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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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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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Nuclear Fast Red for Nuclei

By Dye Type, Hematoxylin Alternatives, Protocols, Stain Type
Protocol

Nuclear Fast Red

for Nuclei

7
steps
5
materials
print

Materials

Solution A

MaterialAmount
Nuclear fast red1g
Potassium aluminum sulphate50g
Distilled water500mL

Preparation

  1. Dissolve the dye and alum into the water.
  2. Boil for 5 minutes. Cool and filter.

Solution B

MaterialAmount
Tartrazine1g
Distilled water1L

Tissue Sample

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

Protocol

  1. Perform the main staining method.
  2. Place into solution A for 5 to 10 minutes
  3. Rinse with water.
  4. Optionally, place into solution B for 30 seconds.
  5. Rinse with distilled water.
  6. Return to the main staining method, or
  7. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  red
  • Cytoplasm  –  yellow or unstained
  • Other tissues  –  according to the main staining method

Safety Note

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

References

  1. Kiernan. J.A., (1999)
    Histological and histochemical methods: Theory and practice, Ed. 3
    Butterworth Heinemann, Oxford, UK.
March 30, 2023
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Papamiltiades’ Alum Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Papamiltiades' Alum Hematoxylin

6
steps
7
materials
print

Materials

MaterialAmountFunction
Hematoxylin4 gDye
Aluminum sulphate10 gMordant
Distilled water900 mLSolvent
Zinc sulphate5 g–
Potassium iodide4 gStabiliser
Glacial acetic acid32 mLAcidifier
Glycerol100 mLStabiliser

Compounding Procedure

  1. Dissolve the hematoxylin in 400 mL water.
  2. Dissolve the aluminum sulphate in 200 mL water.
  3. Dissolve the zinc sulphate in 100 mL water.
  4. Dissolve the potassium iodide in 100 mL water.
  5. Combine the four solutions, then add the acetic acid and glycerol.
  6. The solution may be used immediately, and is stable for about two months.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse with water and blue.
  4. Rinse well with water.
  5. Counterstain if desired.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • The solution is progressive.
  • The purpose of the zinc sulphate and potassium iodide are not clear.
  • The staining time should be determined by trial.
  • Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

Safety Note

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

References

  1. Putt, F.A.
    Manual of Histopathological Staining Methods
    John Wiley & Sons, New York, NY., USA
March 30, 2023
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Paquin & Goddard’s Iron Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Paquin & Goddard's Iron Hematoxylin

8
steps
7
materials
print

Materials

Hematoxylin Solution

MaterialAmountFunction
Hematoxylin0.8 gDye
Ferric ammonium sulfate5 gMordant
Ammonium sulfate0.7 g–
95% ethanol25 mLSolvent
Glycerol13 mLSolvent
Distilled water75 mLSolvent

Picric Ethanol

MaterialAmountFunction
Picric acid, saturated ethanolic.6 mLAcid
95% ethanol94 mLSolvent

Compounding Procedure

  1. Combine the glycerol and ethanol.
  2. Add the hematoxylin and dissolve using gentle heat.
  3. Dissolve the other ingredients in the water.
  4. Add slowly to the hematoxylin solution with agitation.
  5. Let stand for 24 hours before use.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 5 minutes.
  3. Rinse with tap water.
  4. Differentiate briefly in picric ethanol.
  5. Wash well in running tap water to blue.
  6. Rinse with distilled water.
  7. Counterstain if desired.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Background  –  as counterstain or unstained

Notes

  • This was specified as an acid resistant nuclear stain prior to Paquin & Goddard’s trichrome. It is also suitable for other methods requiring an acid resistant nuclear stain.

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:
    Paquin and Goddard, (1947)
    Bulletin of the International Association of Medical Museums
    and Journal of Technical Methods, v. 27, p. 198
March 30, 2023
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Pollak’s Trichrome for Muscle and Collagen

By Protocols, Stain Type, Trichrome Staining, Trichrome, One-Step
Protocol

Pollak's Trichrome

for Muscle and Collagen

6
steps
11
materials
print

Materials

  • Weigert’s iron hematoxylin or equivalent
  • Solution A
    MaterialAmount
    Orange G0.25g
    Ponceau 2R0.33g
    Acid fuchsin0.17g
    Light green SF0.15g
    Phosphotungstic acid0.5g
    Phosphomolybdic acid0.5g
    Acetic acid, glacial1mL
    Ethanol, 95%50mL
    Distilled water50mL
  • Solution B
    MaterialAmount
    Acetic acid, glacial0.2mL
    Distilled water100mL

Preparation of Solution A

  1. Mix the water, ethanol and acetic acid together.
  2. Divide into four, and dissolve as follows:
    1. Phosphomolybdic acid
    2. Phosphotungstic acid and orange G
    3. Light green SF
    4. Ponceau 2R and acid fuchsin
  3. Combine all solutions and filter.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with Weigert’s iron hematoxylin or equivalent.
  3. Place into solution A for 3-7 minute.
  4. Place into solution B until differentiated.
  5. Dehydrate with ethanol.
  6. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Erythrocytes  –  yellow
  • Cytoplasm  –  red
  • Muscle  –  red
  • Collagen  –  blue

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:
    Pollack, (1944)
    Archives of pathology and laboratory medicine, v. 37, pp. 294
    Chicago, USA
March 30, 2023
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Pusey’s Alum Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Pusey's Alum Hematoxylin

6
steps
6
materials
print

This solution is described as a modification of Mayer’s hemalum, but please refer to that formula.

Materials

MaterialAmountFunction
Hematoxylin, aged 10% ethanolic16 mLDye
Ammonium alum60 gMordant
Distilled water1 LSolvent
Sodium iodate0.25 gOxidant
Citric acid, 5% aqueous7 mLAcidifier
Chloral hydrate50 gStabiliser

Compounding Procedure

  1. Dissolve the Alum in the water using low heat, but do not boil.
  2. Add the hematoxylin solution and mix well.
  3. Add the sodium iodate and dissolve.
  4. Leave for 30 minutes.
  5. Add the chloral hydrate and dissolve.
  6. Add the citric acid and mix well.
  7. Check that the pH is 2.45, adjust with citric acid if not.
  8. It may be used immediately.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 5-10 minutes.
  3. Rinse with water and blue.
  4. Rinse well with water.
  5. Counterstain if desired.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • The alcoholic hematoxylin solution should be aged (partially oxidised).
  • The staining time was not given, but 5-10 minutes should suffice.
  • 16 mL of a 10% solution of hematoxylin equates to 1.6 grams dye. With 60 grams of mordant and strong oxidation this would indicate a darkly staining progressive solution.
  • Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

Safety Note

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

References

  1. Pusey’s modified Mayer’s hematoxylin,
    Journal of Histotechnology, v.2, No.2, p.54, 1979
    citing:
    Villaneuva, A.R., (1976)
    Methods of preparing and interpreting mineralized sections of bone, in:
    Proceedings of the First Workshop on Bone Morphometry
    Jaworski, Z. F. G., Editor, (1976).
March 30, 2023
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Rawitz’ Alum Hematoxylin Variants

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Rawitz' Alum Hematoxylin Variants

8
steps
7
materials
print

Materials

MaterialVariantFunction
1895a1895b1909
Hematoxylin10 g––Dye
Hematein–2.5 g2 gDye
Potassium alum10 g––Mordant
Ammonium alum–15 g–Mordant
Aluminum nitrate––20 gMordant
Distilled water650 mL500 mL500 mLSolvent
Glycerol350 mL500 mL500 mLStabiliser

Compounding Procedure

  1. Dissolve the aluminum salt and dye in the water.
  2. When dissolved, add glycerol.
  3. The 1895a formula should be allowed to ripen.
  4. The 1895b and 1909 formulas may be used immediately.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse well with water.
  4. Differentiate with acid ethanol if necessary.
  5. Rinse with water and blue.
  6. Rinse well with water.
  7. Counterstain if desired.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • The 1895b and 1909 formulae use hematein instead of hematoxylin.
  • The Microtomists Formulary and Guide gives the 1895b and 1909 formulae as containing 500 millilitres of glycerol. The Microtomists Vade-Mecum gives these two formulae as containing 500 grams of glycerol. This equates to 400 millilitres based on a specific gravity of 1.25.
  • The staining time should be determined by trial.
  • Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
  • Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate 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.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
  2. Bolles Lee, A.. Edited by Gatenby, J.B. and Beams, H.W., (1950)
    The Microtomist’s Vade-Mecum. 11 ed.,
    Churchill, London, UK.
March 30, 2023
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