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Harris & Power’s Alum Hematoxylin

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

Harris & Power's Alum Hematoxylin

8
steps
4
materials
print

Materials

MaterialAmountFunction
Hematoxylin20 gDye
Potassium alum60 gMordant
Distilled water100 mLSolvent
100% ethanol6 mLSolvent

Compounding Procedure

  1. Mix the Alum and hematoxylin in a mortar.
  2. Add the water, little by little, while grinding the mixture.
  3. Filter, and add the ethanol.

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

  • This very strong mixture should, presumably, be left to ripen for some time.
  • Its characteristics and recommended use were not given.
  • 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.
March 30, 2023
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Haug’s Alum Hematoxylin

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

Haug's Alum Hematoxylin

8
steps
4
materials
print

Materials

MaterialAmountFunction
Hematoxylin5.5 gDye
Aluminum acetate5 gMordant
Distilled water100 mLSolvent
100% ethanol5 mLSolvent

Compounding Procedure

  1. Dissolve the hematoxylin in ethanol.
  2. Dissolve the aluminum acetate in water.
  3. Combine.

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

  • This strong formula should, presumably, be left for some time to ripen.
  • The characteristics and recommended use were not given.
  • 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.
March 30, 2023
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Haythorne’s Trichrome for Muscle and Collagen

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

Haythorne's Trichrome

for Muscle and Collagen

11
steps
10
materials
print

Materials

  • Böhmer’s alum hematoxylin
  • Solution A
    MaterialAmount
    Orange G0.8g
    Ferric ammonium sulphate5g
    Hydrochloric acid0.06mL
    Ethanol, 95%4mL
    Distilled water100mL

    Dissolve the iron alum into 25 mL of the water.

    Combine the remaining ingredients.

    Mix the two solutions together and filter.

  • Solution B
    MaterialAmount
    Acid fuchsin1g
    Distilled water100mL
  • Solution C
    MaterialAmount
    Aniline blue2.5g
    Orange G2.5g
    Phosphomolybdic acidtosaturation
    Distilled water100mL

Tissue Sample

Zenker fixation was recommended, and 5µ paraffin sections would be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixation could probably be used with secondary fixation of sections in Bouin’s fluid.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with Böhmer’s hematoxylin for 30 minutes.
  3. Place into solution A for 2 minutes.
  4. Wash with water for 5 minutes.
  5. Place into solution B for 3 minutes.
  6. Blot.
  7. Place into solution C for 20 minutes.
  8. Blot.
  9. Rinse quickly with 95% ethanol.
  10. Dehydrate and differentiate with absolute ethanol.
  11. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  reddish black
  • Erythrocytes  –  orange
  • Keratin  –  orange
  • Cytoplasm  –  red
  • Collagen  –  blue
  • Cartilage  –  blue

Notes

  • The iron alum in solution A probably serves to mordant the Böhmer’s hematoxylin, converting it into an iron hematoxylin, thus making the nuclear stain acid resistant.
  • Böhmer’s stain is an obsolete alum hematoxylin and a more modern progressive hemalum, such as Mayer’s, may be suitable.
  • Alternatively, Böhmer’s hematoxylin could probably be replaced with an acid resistant nuclear stain such as the celestine blue hemalum sequence, or Weigert’s iron hematoxylin. If so, the iron alum in solution A would likely be redundant (and the method would no longer be Haythorne’s).

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:
    Haythorne, (1916)
    Bulletin of the International Association of Medical Museums
    and Journal of Technical Methods., v. 6, p. 61
    Montreal, Que. Canada & Washington, DC, USA
March 30, 2023
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Heidenhain’s Iron Hematoxylin

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

Heidenhain's Iron Hematoxylin

8
steps
4
materials
print

Materials

Solution A

MaterialAmountFunction
Ferric ammonium sulfate2.5 gMordant
Distilled water100 mLSolvent

Solution

MaterialAmountFunction
Hematoxylin0.5 gDye
Distilled water100 mLSolvent

Compounding Procedure

  1. Make each solution separately.
  2. Solution B should be ripened for a minimum of one month.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into solution A for 30 minutes to 24 hours.
  3. Rinse with distilled water.
  4. Place into solution B for 30 minutes to 24 hours.
  5. Rinse with tap water.
  6. Differentiate in solution A, controlling microscopically.
  7. Wash well in running tap water to blue.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei and other structures  –  blue to black

Notes

  • The stock solutions are stable for some time.
  • The hematoxylin solution needs to be ripened.
  • Staining at elevated temperatures (not over 60°C) will shorten the required times. Always differentiate at room temperature.
  • The degree of differentiation will determine which tissue components are prominent. The method can demonstrate many structures, including chromosomes, nuclear components, mitochondria and muscle striations
  • The solutions may be reused, with the exception of solution A, which is used to differentiate, which should be fresh each time.
  • Counterstaining is not recommended.
  • This method is usually recommended for monochrome photography.
  • Compare Heidenhain’s iron hematoxylin technique to other published variations.

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:
    Heidenhain, M., (1892).
    Festschrift Herrn A. von Kolloker zur Feier seines
    fünfzigjährigen medicinischen
    Doktorjubiläums, p.118.
    Wilhelm Engellmans, Leipzig, Germany
March 30, 2023
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Heidenhain’s Vanadium Hematoxylin

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

Heidenhain’s Vanadium Hematoxylin

4
steps
3
materials
print

Materials

Stock A

MaterialAmountFunction
Hematoxylin0.5 gDye
Distilled water100 mLSolvent

Stock B

MaterialAmountFunction
Ammonium vanadate0.25 gMordant
Distilled water100 mLSolvent

Working Solution

MaterialAmountFunction
Stock A60 mLDye
Stock B30 mLMordant

Compounding Procedure

  1. Make each stock solution separately.
  2. Combine in the specified proportions.

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. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • No details were given.

Notes

  • The staining time should be determined by trial.

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:
    Böhm and Oppel, (1907)
    Manuel de technique microsopique, ed. 4, p. 105.
    Vigot, Paris, France
March 30, 2023
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Held’s Molybdenum Hematoxylin

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

Held's Molybdenum Hematoxylin

6
steps
6
materials
print

Materials

Stock Solution

MaterialVariation IVariation IIFunction
Hematoxylin1 g1 gDye
Phosphomolybdic acid15 g–Mordant
Molybdic acid–excessMordant
Distilled water30 mL30 mLSolvent
95% ethanol70 mL70 mLSolvent

Solution A

MaterialAmountFunction
Ferric ammonium sulfate5 gMordant
Distilled water100 mLSolvent

Solution B

MaterialAmountFunction
Stock solution5 mL–
Distilled water95 mLSolvent

Compounding Procedures

Stock solution

  1. Mix the water and ethanol together.
  2. Dissolve the hematoxylin.
  3. Add the molybdenum compound specified and shake frequently over a period of one month until there is a distinct colour change to a deep blue-black, then decant.
  4. The staining improves with aging.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Optionally, place into solution A for 24 hours.
  3. Place into solution B for 12-24 hours.
  4. Differentiate with solution A if overstained.
  5. Wash in running tap water.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Tissue components  –  various shades.

Notes

  • Variation I is from the Microtomist’s Formulary and Guide and specifies phosphomolybdic acid, variation II is from the Microtomists Vade Mecum and specifies molybdic acid.
  • It is recommended for developing nerve tissues.

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:
    Bolles-Lee, A.; Edited by Gatenby, J. B. & Painter, T. S., (1937)
    The microtomists’s Vade-Mecum, p. 374
    Blakiston, Philadelphia, USA
  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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Slidders’ Fuchsin-Miller for Fibrin

By Fibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Yellowsolve Staining
Protocol

Slidders’ Fuchsin-Miller

for Fibrin

16
steps
7
materials
print

Materials

  • An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
  • Fuchsin
    MaterialAmount
    Acid fuchsin1g
    Acetic acid, glacial2.5mL
    Distilled water100mL
  • Miller
    MaterialAmount
    Milling yellow 3G2.5g
    2-ethoxy-ethanol100mL
  • Phosphotungstic acid
    MaterialAmount
    Phosphotungstic acid1g
    Distilled water100mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate or B5 overnight. Paraffin process overnight. Overnight formalin fixation and paraffin processing can produce acceptable results if sections are refixed for an hour in Bouin’s picro-acetic-formalin mixture at 56°C. Avoid rapid fixation with formalin and short processing, as this produces tissues that stain poorly even with the secondary fixation specified. Optimal results are obtained with extended mercuric chloride fixation, thorough processing, degreasing and secondary fixation of sections. Sections should be 3-5 µ thick.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. If mercury fixed, remove with the iodine-thiosulphate sequence.
  3. If not mercury fixed, place into Bouin’s fluid at 56°C for 1 hour.
  4. Rinse well with distilled water.
  5. Stain nuclei with an acid resistant nuclear stain.
  6. Rinse with 95% ethanol.
  7. Place in fuchsin for 10 minutes.
  8. Rinse with distilled water.
  9. Differentiate with phosphotungstic acid for 5 minutes.
  10. Rinse well with distilled water.
  11. Blot.
  12. Rinse well with cellosolve.
  13. Place into milling yellow until fibrin is red and tissues are yellow.
    This step takes from ½ – 4 hours.
  14. Rinse briefly with 1% aqueous acetic acid.
  15. Rinse with cellosolve.
  16. Clear with xylene and mount with a resinous medium.

Expected Results

  • Fibrin  –  red
  • Other tissue  –  yellow
  • Nuclei  –  black

Notes

  • It is very important that the milling yellow solution be completely anhydrous. Even small amounts of water or other solvents can cause problems with displacement and incomplete removal of red dye. For that reason, step 12 should be thorough and the milling yellow should be used in a Coplin jar with a lid sealed with tape.
  • A well stained section would show red fibrin only, muscle and erythrocytes being yellow. With poorly fixed material, both erythrocytes and muscle fibres may resist displacement of the red dye and they may have some residual red colouring. Sometimes this may be overcome by prestaining either with saturated picric acid in absolute ethanol or the MSB martius yellow solution for two minutes immediately before step 7, but a better resolution is correct fixation and processing.
  • Some intracellular materials may stain red, such as Paneth cell granules.

Safety Note

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

References

  1. Drury, R.A.B. and Wallington, E.A., (1980)
    Carleton’s histological technique Ed. 5
    Oxford University Press, Oxford, UK.
  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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Martius, Scarlet and Blue (MSB) for Fibrin

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

Martius, Scarlet and Blue (MSB)

for Fibrin

14
steps
8
materials
print

The MSB (Martius, Scarlet and Blue) method for fibrin is a reliable technique. It is more automatic than other methods, i.e. it is less dependent on skill and experience and is consequently suitable for a routine laboratory. Overnight mercuric chloride fixation (formol sublimate or B5) is preferred, followed by overnight paraffin processing, although formalin fixed, paraffin embedded material can produce acceptable results if sections are refixed for an hour in Bouin’s picro-acetic-formalin mixture at 56°C. Optimal results are obtained with extended mercuric chloride fixation, thorough processing, degreasing and secondary fixation of sections, as for the Picro-Mallory.

Materials

  • An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
  • Martius
    MaterialAmount
    Martius yellow0.5g
    Phosphotungstic acid2g
    Ethanol, 95%100mL
  • Scarlet
    MaterialAmount
    Crystal scarlet1g
    Acetic acid, glacial2.5mL
    Distilled water97.5mL
  • Blue
    MaterialAmount
    Methyl blue0.5g
    Acetic acid, glacial1mL
    Distilled water98mL
  • Phosphotungstic acid
    MaterialAmount
    Phosphotungstic acid1g
    Distilled water100mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate (or B5) overnight. Paraffin process overnight. Overnight formalin fixation is usually satisfactory, but avoid rapid fixation with formalin and short processing, as this produces tissues that stain poorly even with the secondary fixation specified. Sections should be 3-5 µ thick.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. If mercury fixed, remove with the iodine-thiosulphate sequence.
  3. If not mercury fixed, place into Bouin’s fluid at 56°C for 1 hour.
  4. Rinse well in distilled water.
  5. Stain nuclei with an acid resistant nuclear stain.
  6. Rinse with 95% ethanol.
  7. Place in martius yellow for 2 minutes.
  8. Rinse with distilled water.
  9. Place in crystal scarlet for 10 minutes.
  10. Differentiate with phosphotungstic acid until only fibrin is red (up to 10 minutes).
  11. Place in methyl blue until collagen is blue (up to 10 minutes).
  12. Rinse briefly with 1% aqueous acetic acid.
  13. Dehydrate rapidly with ethanol.
  14. Clear with xylene and mount with a resinous medium.

Expected Results

  • Fibrin  –  red
  • Fresh fibrin  –  yellow
  • Erythrocytes  –  yellow
  • Connective tissue  –  blue

Notes

  • Crystal scarlet is more commonly known as ponceau 6R.
  • Steps 10 & 11 specify the time as up to. These times should be established by inspection, but will generally remain consistent.
  • Lendrum’s recommendation for formalin fixed material was to dewax sections and treat with trichlorethylene in a sealed contained for 48 hours at 56°C, then to refix in absolute ethanol saturated with both picric acid and mercuric chloride for 24 hours before proceeding to step 2. The alternative treatment with Bouin’s fluid given at step 3 is often satisfactory.
  • Bancroft notes that dyes other than those originally given may be used. Some may not be easily available, and no CI numbers were given.
    ColorDye Options
    YellowLissamine fast yellow
    BlueDurazol blue
    Pontamine sky blue
    Fast green FCF
    Naphthalene black 10B
    RedPonceau de xylidine
    Azofuchsin

Safety Note

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

References

  1. Drury, R.A.B. and Wallington, E.A., (1980)
    Carleton’s histological technique Ed. 5
    Oxford University Press, Oxford, UK.
  2. Culling C.F.A., (1974)
    Handbook of histopathological and histochemical techniques Ed. 3
    Butterworth, London, UK.
  3. 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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Obadiah for Fibrin

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

Obadiah

for Fibrin

14
steps
11
materials
print

The name of this stain comes from the letters OBDR45, which refer to the dyes used: Orange, Blue, Direct Red 45 (a synonym for one of the red dyes).

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 acidtosaturation
    Mercuric chloridetosaturation
  • Orange
    MaterialAmount
    Orange G0.5g
    Phosphotungstic acid1g
    Ethanol, 95%100mL
  • Blue
    MaterialAmount
    Naphthalene blue black CS1g
    Acetic acid, glacial1mL
    Distilled water99mL
  • Red – Option 1
    MaterialAmount
    Chicago red2.5g
    Acetic acid, glacial2.5mL
    Distilled water97.5mL
  • Red – Option 2
    MaterialAmount
    Polar brilliant red BN1g
    Acetic acid, glacial1mL
    Distilled water99mL

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 orange stain for 2 minutes.
  8. Rinse with distilled water.
  9. Place in the Blue stain up to 30 minutes.
  10. Differentiate with the polyacid for 5 minutes.
  11. Place in the Red stain 15-20 minutes (polar brilliant red) or 15-30 minutes (chicago red).
  12. Rinse with distilled water.
  13. Dehydrate with ethanol.
  14. Clear with xylene and mount with a resinous medium.

Expected Results

  • Old fibrin  –  black
  • Younger fibrin  –  may be yellow
  • Connective tissue  –  red

Notes

  • Note that this method reverses the usual order and uses a blue stain before the red stain.
  • Lendrum considered that this method demonstrated fibrin that other methods stained as collagen.

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.
March 30, 2023
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Picro-Mallory for Fibrin – Long Version

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

Picro-Mallory

for Fibrin – Long Version

15
steps
12
materials
print

Lendrum, Fraser and others published several fibrin stains, the most well known being the Picro-Mallory. There are several variants of the method, some more complicated than others.

See this alternative protocol for a shorter version.

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. Adequate results can be obtained with thorough formalin fixation (24 hours), thorough processing (overnight), sectioning, followed by refixing in Bouin’s fluid at 56°C for an hour or so. However, results are inferior to those obtained by the full procedure. It is also a method where experience is required for optimal results.

Materials

  • An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the
    celestine blue-hemalum sequence.
  • Yellow mordant
    MaterialAmount
    Picric acid, sat. in 80% ethanol100mL
    Orange G0.2g
    Lissamine yellow0.2g
  • Stock differentiator
    MaterialAmount
    Picric acid2.5g
    Ethanol, 95%100mL
    Phosphotungstic acid25g

    Dissolve ingredients and filter.

  • Red differentiator
    MaterialAmount
    Stock differentiator40mL
    Ethanol, 95%20mL
    Distilled water90mL
  • Blue differentiator
    MaterialAmount
    Stock differentiator10mL
    Distilled water90mL
  • Red stain
    MaterialOption 1Option 2Option 3
    Acid fuchsin1g–0.4g
    Lissamine fast red––0.2g
    Biebrich scarlet–1g–
    Acetic acid, glacial1mL1mL1mL
    Distilled water99mL99mL99mL
  • Blue stain
    MaterialAmount
    Methyl blue1g
    Acetic acid, glacial1mL
    Distilled water99mL

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, especially with formalin fixatives, as this produces tissues that stain poorly even with secondary fixation such as Bouin’s fluid at 56°C for an hour. Sections should be 3-5 µ thick.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Remove mercury pigment with the iodine-thiosulphate sequence.
  3. Stain nuclei with an acid resistant nuclear stain.
  4. Place in yellow mordant for 2-3 minutes.
  5. Wash in distilled water until only erythrocytes are yellow.
  6. Place in the red stain for 5-10 minutes.
  7. Rinse with 1% aqueous acetic acid.
  8. Differentiate with the red differentiator until fibrin is prominent microscopically.
  9. Rinse well in distilled water.
  10. Place in the blue stain for 5 minutes.
  11. Rinse briefly with 1% aqueous acetic acid.
  12. Place in the blue differentiator for 1-2 minutes.
  13. Biefly rinse with 1% aqueous acetic acid.
  14. Dehydrate with ethanol.
  15. Clear with xylene and mount with a resinous medium.

Expected Results

  • Fibrin  –  clear red
  • Erythrocytes  –  yellow
  • Connective tissue  –  blue

Notes

  • The most common dye combination is acid fuchsin and methyl blue.
  • Adequate results may be obtained with 24-48 hours fixation in neutral buffered formalin, overnight paraffin processing and secondary fixation of sections with Bouin’s picro-formol-acetic for 1 hour at 56°C.
  • Good results are obtained with the fixation and processing recommended in the text.
  • Optimal results are obtained with the fixation and processing recommended in the text, followed by degreasing and secondary fixation in picro-mercuric ethanol.

    Replace step 1 with the following:

    1. Dewax sections with xylene.
    2. Place in trichlorethylene in a sealed container at 56°C for 48 hours.
    3. Rinse sections well with absolute ethanol.
    4. Place in picro-mercuric-ethanol for 24 hours.
    5. Wash sections with water, and continue from step 2.

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.
March 30, 2023
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