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Yellowsolve II for Cellular Inclusions

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

Yellowsolve II

for Cellular Inclusions

10
steps
7
materials
print

This method is also known as the Rhoda-Coomassie.

Materials

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are likely suitable. It should be noted, however, that the authors of this method favoured extended fixation in formal sublimate of up to 10 days for fibrin. This fixative is now deprecated due to its mercuric chloride content. With other staining methods, pretreatment of sections with Bouin’s fluid for an hour at 60°C can compensate for the lack of mercury fixation to a large degree.

Protocol

  1. Dewax sections with xylene.
  2. Place in trichlorethylene for 24 to 48 hours.
  3. Bring sections to water.
  4. Stain nuclei with the celestine blue-hemalum sequence.
  5. Wash well with water.
  6. Place in solution A for 5 minutes.
  7. Rinse with 2-ethoxyethanol.
  8. Differentiate with solution B, controlling microscopically.
  9. Rinse with 2-ethoxyethanol.
  10. Clear in xylene and mount with a synthetic resinous medium.

Expected Results

  • Cell inclusions  –  red
  • Background  –  yellow
  • Nuclei  –  black

Notes

  • Inclusions stained may include mast cells, plasma cells and Paneth cells as well as fibrin.
  • 2-Ethoxyethanol is also known as cellosolve or ethylene glycol monoethyl ether, and has the formula CH3CH2OCH2CH2OH.
  • Trichlorethylene has the formula ClHC=CCl2 or C2HCl3
  • Trichlorethylene should be used in a fume hood.

Safety Note

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

References

  1. Lendrum A C, Fraser D S, Slidders W and Henderson R. (1962)
    Studies on the character and staining of fibrin.
    Journal of clinical pathology, v. 15, p. 401.
March 30, 2023
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Einarson’s Gallocyanin-Chrome Alum

By Intracytoplasmic Granules, Nissl Bodies, Plasma Cells, Protocols, Stain Target
Protocol

Einarson’s

Gallocyanin-Chrome Alum

5
steps
3
materials
print

Materials

Solution

MaterialAmount
Chrome alum5g
Gallocyanin0.15g
Distilled water100mL

Solution Preparation

  1. Add the chrome alum and gallocyanin to the water.
  2. Bring to a boil and simmer for 20 minutes.
  3. Cool and filter.

Tissue Sample

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

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place in the staining solution for 24-48 hours.
  3. Rinse well with water.
  4. Dehydrate with ethanols.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nucleic acids  –  blue

Notes

  • Chrome alum is chromium potassium sulfate dodecahydrate, CrK(SO4)2·12H2O
  • Culling states that the pH is 1.64, and that changing this will eliminate or accentuate non-specific staining. Addition of up to 10 mL N hydrochloric acid will eliminate background staining, and addition of up to 5 mL N sodium hydroxide will accentuate it. In either case, staining of nucleic acids is not affected.

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:
    Einarson, (1932)
    American Journal of Pathology, v. 8, p. 295
    Boston, USA
  2. Culling C.F.A., (1974)
    Handbook of histopathological and histochemical techniques Ed. 3
    Butterworth, London, UK.
March 30, 2023
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Cole’s Alum Hematoxylin Variants

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

Cole's Alum Hematoxylin Variants

8
steps
7
materials
print

References to Cole’s hematoxylin that do not specify which of the two formulae is meant, usually refer to the 1943 formula.

Materials

MaterialVariantFunction
19031943
Hematoxylin6 g1.5 gDye
Ammonium alum6 g100 gMordant
Distilled water320 mL950 mLSolvent
100% ethanol320 mL50 mLSolvent
Glycerol290 mL–Stabiliser
Iodine–0.5 gOxidant
Glacial acetic acid75 mLSee noteAcidifier

Compounding Procedures

1903

  1. Dissolve the alum in water.
  2. Dissolve the hematoxylin in ethanol.
  3. Combine, then add the other ingredients and mix well.
  4. The solution must ripen before use.

1943

  1. Dissolve the hematoxylin in 250 mL water with heat.
  2. Dissolve the alum in 700 mL water.
  3. Dissolve the iodine in the ethanol.
  4. Combine the dye and iodine solutions.
  5. Add the alum solution.
  6. Bring to a boil.
  7. Cool and filter.
  8. The solution 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 original 1943 formula contains no added acid. Used progressively, and applied for 5-10 minutes, it gives results similar to those obtained with a differentiated regressive formula.
  • The addition of 20 mL glacial acetic acid to the 1943 formula increases nuclear selectivity and extends the working life of the solution. This is often used for progressive nuclear counterstaining and in the celestine blue-hemalum sequence.
  • The staining time for the 1903 formula 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. Culling, C.F.A., Allison, R.T. and Barr, W.T.
    Cellular Pathology Technique, Ed.4.
    Butterworth, London, UK.
  2. Drury, R.A.B. and Wallington, E.A., (1980)
    Carleton’s histological technique Ed. 5
    Oxford University Press, Oxford, UK.
  3. Bancroft, J.D. and Stevens A. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
  4. 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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Herovici’s Stain for Young and Mature Collagen

By Dye Type, Picro-Fuchsin, Protocols, Stain Type, Trichrome Staining
Protocol

Herovici's Stain

for Young and Mature Collagen

7
steps
7
materials
print

Materials

Tissue Sample

Paraffin sections at 5µ of formol-acetic-ethanol (10:5:85) fixed tissue was recommended.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with the acid resistant nuclear stain.
  3. Rinse well with tap water.
  4. Place into the staining solution for 2 minutes.
  5. Wash with 1% acetic acid for 2 minutes.
  6. Dehydrate with ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Young collagen and reticulin – blue
  • Mature collagen – red
  • Cytoplasm – yellow
  • Nuclei – black

Notes

  • Solution A is van Gieson’s solution.
  • Cook notes that the original included a final step with metanil yellow for cytoplasmic staining, which he omitted.

Safety Note

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

References

  1. Cook, H. C., (1974)
    Manual of Histological Demonstration Techniques
    Butterworths, London, UK.
    Citing:
    Herovici, c., (1963)
    Stain Technology, v. 38, p. 204
March 30, 2023
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Puchtler’s Picro-Sirius red for Collagen

By Dye Type, Picro-Fuchsin, Protocols, Stain Type, Trichrome Staining
Protocol

Puchtler's Picro-Sirius red

for Collagen

6
steps
4
materials
print

Materials

Picro-sirius red

MaterialAmount
Sirius red F3B0.5g
Saturated aqueous picric acid500mL

Acetic acid water

MaterialAmount
Acetic acid, glacial5mL
Distilled water11mL

Tissue Sample

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

Protocol

  1. Bring sections to water.
  2. Optionally, stain nuclei with an acid resistant nuclear stain.
  3. Wash well with water.
  4. Place into Picro-sirius red solution for 1 hour.
  5. Wash with two changes of acetic acid water.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Light Microscopy

  • Nuclei – black if stained
  • Collagen – red
  • Cytoplasm – yellow

Polarising Microscopy

  • Large fibres – yellow or orange birefringence
  • Thin fibres – green birefringence

Notes

  • This method can be used as an alternative for van Gieson’s stain or, using polarising microscopy, as a sensitive method for collagen.

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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van Gieson’s Stain for Collagen

By Dye Type, Picro-Fuchsin, Protocols, Stain Type, Trichrome Staining
Protocol

van Gieson's Stain

for Collagen

7
steps
3
materials
print

Materials

Tissue Sample

Paraffin sections at 5µ are suitable. Many fixatives, including formalin, are satisfactory. Stains using acid dyes often benefit from picric acid or mercuric chloride fixation.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with the acid resistant nuclear stain.
  3. Rinse well with tap water.
  4. Place into the staining solution for 2-5 minutes.
  5. Optionally, rinse quickly with distilled water.
  6. Dehydrate with ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Collagen – red
  • Cytoplasm – yellow
  • Nuclei – black

Notes

  • This method is often used to counterstain other primary staining methods. In that case the nuclear stain may not be necessary and should be ommitted.

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. Drury, R.A.B. and Wallington, E.A., (1980)
    Carleton’s histological technique Ed. 5
    Oxford University Press, Oxford, UK.
  3. Culling C.F.A., (1974)
    Handbook of histopathological and histochemical techniques Ed. 3
    Butterworth, London, UK.
March 30, 2023
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Kohashi’s Trichrome for Elastic and Collagen

By Dye Type, Elastic Fibers, Orcein, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, Multi-Step
Protocol

Kohashi's Trichrome

for Elastic and Collagen

12
steps
14
materials
print

Materials

Solution A

MaterialAmount
Azocarmine0.1g
Acetic acid, glacial1mL
Distilled water99mL

Solution B

MaterialAmount
Aniline0.1mL
Ethanol, 95%100mL

Solution C

MaterialAmount
Acetic acid, glacial1mL
Ethanol, 95%100mL

Solution D

MaterialAmount
Phosphotungstic acid5g
Distilled water100mL

Solution E

MaterialAmount
Eosin B0.7g
Acid fuchsin, sat. aqu.4mL
Unna’s elastic stain35mL
Ethanol, 50%35mL
Glycerol40mL

Tissue Sample

Many fixatives are satisfactory. 5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. 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. Place into solution A for 12-15 minutes.
  3. Rinse quickly with distilled water.
  4. Differentiate nuclei with solution B.
  5. Place into solution C for 30-60 seconds.
  6. Rinse with distilled water.
  7. Place into solution D for 30-60 minutes.
  8. Rinse with distilled water.
  9. Place into solution E for 15-20 minutes.
  10. Place into 95% ethanol until differentiated.
  11. Dehydrate with ethanol.
  12. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  red
  • Erythrocytes  –  orange
  • Elastic fibres  –  purple
  • Cytoplasm  –  red
  • Collagen  –  blue

Notes

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:
    Kohashi, (1937)
    Folia anatomica Japonica, v.15, pp.175
March 30, 2023
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Mollier’s trichrome for Elastic and Collagen

By Dye Type, Elastic Fibers, Orcein, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, Multi-Step
Protocol

Mollier's trichrome

for Elastic and Collagen

13
steps
13
materials
print

Materials

  • Weigert’s iron hematoxylin or equivalent
  • Solution A
    MaterialAmount
    Orcein0.8g
    Hydrochloric acid1mL
    Ethanol, 100%50mL
    Distilled water50mL
  • Solution B
    MaterialAmount
    Azocarmine2g
    Acetic acid, glacial1mL
    Distilled water100mL
  • Solution C
    MaterialAmount
    Phosphotungstic acid5g
    Distilled water100mL
  • Solution D
    MaterialAmount
    Naphthol green B1g
    Acetic acid, glacial1mL
    Distilled water100mL

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. Place into solution A for 12 hours.
  3. Stain nuclei with Weigert’s iron hematoxylin for 1-3 minutes.
  4. Differentiate the nuclear stain if necessary.
  5. Wash with water for 15 minutes.
  6. Place into solution B for 15-30 minutes.
  7. Rinse with distilled water.
  8. decolorise in solution C for 2-6 hours, changing the solution three times.
  9. Rinse quickly with distilled water.
  10. Place into solution D for 15-30 minutes.
  11. Agitate vigorously in 95% ethanol for 30 seconds.
  12. Dehydrate with ethanol.
  13. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Elastic  –  black
  • Erythrocytes  –  red
  • Cytoplasm  –  purple
  • Collagen  –  green

Notes

  • Solution A is the Unna-Taenzer elastic solution.

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:
    Mollier, (1938)
    Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik, v.55, pp.472.
    And:
    von Kahlden, C. and Laurent, O., (1896)
    Technique microscopique, pp.143
    Carré, Paris, France
March 30, 2023
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Roque’s Stain for Cell Inclusions

By Intracytoplasmic Granules, Plasma Cells, Protocols, Stain Target
Protocol

Roque's Stain

for Cell Inclusions

7
steps
6
materials
print

Materials

Stock solution A

MaterialAmount
Citrate buffer, pH 5.8100mL
Methyl green, purified0.1g
Thionin0.0165g

Dissolve the thionin in a small amount of water. Add the buffer and methyl green. Shake and filter. Use fresh.

Citrate buffer, pH 5.8

MaterialAmount
Hydrochloric acid, 0.01M42mL
Sodium citrate, 0.01M58mL

Dehydrant

MaterialAmount
Tertiary butanol80mL
Ethanol, absolute20mL

Tissue Sample

Fix 2mm thick pieces of tissue in 10% formalin containing 1% sodium acetate for 3 hours. Fix smears with methanol.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into the staining solution for 30 minutes at 40°C.
  3. Rinse briefly with distilled water.
  4. Place into dehydrant for 30 seconds
  5. Replace dehydrant, 2 changes, 3 minutes each.
  6. Rinse with absolute ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue-green
  • Nucleolar and cytoplasmic basophil substances  –  red-purple

Notes

  • The reference specifies methyl green, but gives the CI number for ethyl green.
  • The methyl green should be purified, but as a powder rather than as a solution:
    • Add about 10g methyl green to 200 mL chloroform in an Erlenmeyer flask.
    • Shake well.
    • Filter under vacuum and in a fume chamber.
    • Repeat until the chloroform is blue-green instead of violet.

Safety Note

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

References

  1. Humason, G. L., (1967).
    Animal Tissue Techniques., pp. 278
    W. H. Freeman and Company, San Francisco, CA, USA.
March 30, 2023
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Bauer Reaction for Carbohydrates

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

Bauer Reaction

for Carbohydrates

9
steps
7
materials
print

Materials

  • A Schiff reagent
  • A progressive hemalum, such as Mayer
  • Chromic acid
    MaterialAmount
    Chromium trioxide4g
    Distilled water100mL
  • Sulfurous acid
    MaterialAmount
    Sodium metabisulfite, 10% aqu.6mL
    Hydrochloric acid, 1N5mL
    Distilled water100mL

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. Oxidize in chromic acid for 40-60 minutes.
  3. Rinse with tap, then distilled water.
  4. Place into Schiff’s reagent for 15 minutes.
  5. Place into sulfurous acid rinses, 3 changes of 2 minutes each.
  6. Wash with running tap water.
  7. Counterstain with hemalum for 1 minute, and blue
  8. Dehydrate with ethanols.
  9. Clear with xylene and mount with a resinous medium.

Expected Results

  • Glycogen, mucin  –  red
  • Fungi  –  red
  • Nuclei  –  blue

Notes

  • Modern practice is to leave out the sulfite rinses and wash with large amounts of tap water.
  • A progressive hemalum should be used as counterstain because regressive hemalums sometimes stain mucin.
  • Mucins are not usually as dark as with a PAS.
  • Applying chromic acid for too long weakens staining due to continued oxidation of the aldehydes first produced.

Safety Note

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

References

  1. McManus, J. F. A. and Mowry, R. W., (1960)
    Staining Methods Histologic and Histochemical
    Harper & Row, New York, NY, USA.
March 30, 2023
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