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Stain Type

Unna’s Alum Hematoxylin

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

Unna's Alum Hematoxylin

8
steps
5
materials
print

Materials

MaterialAmountFunction
Hematoxylin3 gDye
Ammonium alum300 gMordant
Distilled water600 mLSolvent
100% ethanol300 mLSolvent
Sublimed sulfur6 gStabiliser

Compounding Procedure

  1. Dissolve the hematoxylin in the alcohol.
  2. Dissolve the Alum in the water.
  3. Combine the two solutions.
  4. Leave the solution at room temperature to ripen (days).
  5. Add the sulfur and mix well.

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 sulfur is added after the solution has properly ripened.
  • Unna said that the sulfur stabilized the solution in the oxidized state for some time. Others considered glycerol to be superior for this purpose.
  • Ammonium Alum dissolves in water at the rate of about one gram in 7 mL, and it is almost insoluble in ethanol. The formula should therefore require about 90 grams, so the amount specified is in considerable excess.
  • 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.
    Citing:
    Unna, (1892)
    Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik, v.8, p.438
    Leipzig
March 30, 2023
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Wallart & Honette’s Trichrome for Connective Tissues

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

Wallart & Honette's Trichrome

for Connective Tissues

9
steps
6
materials
print

Materials

Stock solution 1

MaterialAmount
Acid fuchsin1g
Acetic acid1mL
Distilled water100mL

Stock solution 2

MaterialAmount
Fast yellow3g
Acetic acid1mL
Distilled water100mL

Stock solution 3

MaterialAmount
Phosphomolybdic acid1g
Distilled water100mL

Working solution A

MaterialAmount
Stock solution 130mL
Stock solution 230mL
Stock solution 330mL

Working solution B

MaterialAmount
Acetic acid1mL
Distilled water100mL

Working solution C

MaterialAmount
Acetic acid1mL
Ethanol 100%100mL

Tissue Sample

5µ paraffin sections of formalin fixed tissue are suitable. Many 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 an acid resistant nuclear stain.
  3. Rinse well with tap water.
  4. Place into solution A for 5 minute.
  5. Rinse quickly with distilled water.
  6. Place into solution B for 5 minutes.
  7. With a pipette, drop solution C onto the slide for 30 seconds.
  8. Dehydrate with ethanol.
  9. Clear with xylene and mount with a resinous medium.

Expected Results

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

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:
    Wallart and Honette, (1934)
    Bulletin d’histologie appliquée à la physiologie et à la pathologie et de technique microscopique, v. 10, p. 404.
March 30, 2023
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Walter’s Trichrome for Elastic and Collagen

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

Walter's Trichrome

for Elastic and Collagen

9
steps
8
materials
print

Materials

Solution A

MaterialAmount
Ferric ammonium sulphate2.5g
Distilled water100mL

Solution B

MaterialAmount
Phosphotungstic acid2g
Distilled water100mL

Solution C

MaterialAmount
Eosin B0.7g
Acid fuchsin, sat. aqu4mL
Unna’s elastic stain35mL
Ethanol, 50%35mL
Glycerol40mL

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 24 hours.
  3. Rinse quickly with distilled water.
  4. Place into solution B for 10 minutes.
  5. Rinse quickly with distilled water.
  6. Place into solution C for 15-20 minutes.
  7. Rinse quickly with 95% ethanol until clouds of dye stop being extracted.
  8. Dehydrate with 100% ethanol for 1 minute.
  9. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  purple
  • Elastic fibres  –  purple
  • Erythrocytes  –  orange
  • 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:
    Walter, (1930)
    Zeitschrift für wissenschaftliche Mikroskopie und für mikroskipische Technik,
    v. 46, pp. 458
March 30, 2023
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Watson’s Alum Hematoxylin

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

Watson's Alum Hematoxylin

8
steps
7
materials
print

Materials

MaterialAmountFunction
Hematoxylin6 gDye
Ammonium alum6 gMordant
Distilled water300 mLSolvent
100% ethanol300 mLSolvent
Glycerol300 mLStabiliser
Glacial acetic acid30 mLAcidifier
Potassium permanganate or

Chloramine T or

Barium hydroxide

0.3 g

3.9 g

6 g

Oxidants

Compounding Procedure

  1. Dissolve the Alum in the water.
  2. If using potassium permanganate, add to the Alum solution.
  3. Dissolve the hematoxylin in ethanol.
  4. Combine the two solutions.
  5. Add the glycerol.
  6. Add the acetic acid (30 mL, unless using barium hydroxide, then add 120 mL)
  7. If using chloramine T or barium hydroxide, add to the solution.

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

  • Use only one of the oxidants named.
  • This is a modification of Ehrlich’s hematoxylin, and is said to be as effective. It was designed to be used immediately after preparation, eliminating the months required for atmospheric oxidation.
  • Gray gives a similar formula for Watson’s hemalum with potassium permanganate, but specifies only 0.6 grams alum. This may be an error.
  • 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. Bolles Lee, A.. Edited by Gatenby, J.B. and Beams, H.W., (1950)
    The Microtomist’s Vade-Mecum. 11 ed.,
    Churchill, London, UK.
    Citing:
    Watson, (1943)
    Journal of the Royal Microscopical Society, v. 63, p. 20
  2. 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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Weigert’s Iron Hematoxylin

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

Weigert's Iron Hematoxylin

8
steps
8
materials
print

Materials

Solution A

MaterialVariantFunction
19031904
Ferric chloride0.4 g0.6 gMordant
Distilled water100 mL100 mLSolvent
Hydrochloric acid–0.75 mLSolvent

Solution B

MaterialVariantFunction
19031904
Hematoxylin1 g1 gDye
95% ethanol100 mL100 mLSolvent

Solution C

MaterialVariantFunction
19031904
Potassium ferricyanide2.5 g–Bleach
Sodium borate2 g–Alkaliniser
Distilled water100 mL–Solvent

Note: The 1904 formula is the solution usually meant when Weigert’s iron hematoxylin is specified as an acid resistant nuclear stain.

Compounding Procedure

For both variants:

  1. Make each solution separately.
  2. Filter.
  3. Immediately before use, combine equal parts of solutions A and B.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 1-2 hours.
  3. Rinse with tap water.
  4. 1903 – Place in solution C until differentiated.
    1904 – Dip briefly in 1% hydrochloric acid in 70% 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

  • The stock solutions are stable for some time.
  • The working solution should be made fresh.
  • The alcoholic hematoxylin solution was originally made by diluting a stock solution of 10% hematoxylin in 95% ethanol.
  • Solution A of the 1904 variant was originally made from a commercial ferric chloride solution, the amounts above being given by Gray based on its formula. The following is now usually specified:30% aqueous ferric chloride – 4 mL

    Distilled water – 100 mL

    Hydrochloric acid – 1 mL

  • Both methods were originally intended to be used without a counterstain for demonstrating chromatin and other structures usually stained by iron hematoxylin. If the 1904 formula is used for this purpose, the staining time should be increased and the excess stain removed with acid ethanol.

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:–
    Ehrlich, P., Krause, R. et. al., (1910)
    Enzyklopädie der mikroskopischen technik, ed. 2, v. 1, p. 231.
    Weigert, K., (1904)
    Zeitschrift für wissenschaftliche mikroskopie und für mikroskopische technik,
    v. 21, p. 1.
  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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Weiss’ Trichrome for Muscle and Collagen

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

Weiss' Trichrome

for Muscle and Collagen

8
steps
7
materials
print

Materials

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 with an acid resistant nuclear stain.
  3. Place into solution A for 1 minute.
  4. Drain.
  5. Place into solution B for 4 minutes.
  6. Wash briefly with distilled water.
  7. Dehydrate with ethanol.
  8. Clear with xylene and mount with a resinous medium.

Expected Results

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

Notes

  • The original recommended nuclear staining in Delafield’s alum hematoxylin, with blueing.
  • This differs from Brillmeyer’s trichrome by using a weaker acid fuchsin solution and staining in aniline blue for a much shorter time.

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:
    Weiss, (1932)
    Stain Technology, v. 7, pp. 131
March 30, 2023
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Yasvoyn’s Iron Hematoxylin

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

Yasvoyn's Iron Hematoxylin

5
steps
3
materials
print

Materials

Solution A

MaterialAmountFunction
Hematoxylin0.1 gDye
Distilled watermLSolvent

Solution B

MaterialAmountFunction
Ferric ammonium sulfate2.5 gMordant
Distilled water100 mLSolvent

Compounding Procedure

  1. Make each solution separately.
  2. For use, add solution B drop by drop to 20 drops of solution A until it just remains blue .
  3. The working solution may be used immediately, but is not stable for long.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 2-5 minutes.
  3. Rinse with water.
  4. Remove excess stain with 70% ethanol if necessary.
  5. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Background  –  as counterstain or unstained

Notes

  • The stock solutions are stable for some time.
  • The working solution should be made fresh.
  • Although not specified, a tap water wash to blue the hematoxylin may be useful following step 4.
  • Following a wash to blue the nuclei, a counterstain could probably be applied, if wished, before dehydration.

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:
    Roskin, G.E, (1946).
    Mikroskopecheskaya technica, p. 150
March 30, 2023
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Gomori’s Impregnation for Reticulin

By Metal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type
Protocol

Gomori's Impregnation

for Reticulin

19
steps
9
materials
print

Materials

  • Silver nitrate, 2% aqu.
  • Silver nitrate, 20% aqu.
  • Strong ammonium hydroxide (s.g. 0.88).
  • Sodium hydroxide, 40% aqu.
  • Periodic acid, 0.5% aqu.
  • Formalin, 3% aqu.
  • Yellow gold chloride, 0.5% aqu.
  • Sodium thiosulphate, 5% aqu.
  • Neutral red, 1% aqu.

Preparation of Ammoniacal Silver

  1. Place 10 mL of 10% silver nitrate in a flask.
  2. Add 2.5 mL of 10% potassium hydroxide.
  3. Allow the precipitate to settle then remove the supernatent with a Pasteur pipette.
  4. Wash the precipitate twice with distilled water, allowing the precipitate to settle and draining after each.
  5. Add 10 mL distilled water.
  6. While swirling, slowly add drops of strong ammonium hydroxide until the precipitate just redissolves.
  7. Slowly add a drop or more of 10% silver nitrate until the solution becomes very faintly opalescent.
  8. Make up to 20 mL with distilled water.

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Oxidise with 1% potassium permanganate for 2 minutes.
  3. Bleach in Oxalic acid for a 2 minutes.
  4. Rinse well with tap water.
  5. Sensitise with 2.5% iron alum solution for 1 minute.
  6. Rinse with tap water.
  7. Rinse well with distilled water.
  8. Treat with ammoniacal silver for 1 to 3 minutes.
  9. Rinse briefly with distilled water.
  10. Reduce in 10% formalin for 3 minutes.
  11. Rinse well with tap water.
  12. Rinse with distilled water.
  13. Tone with 0.2% gold chloride solution.
  14. Rinse with distilled water.
  15. Fix in 5% sodium thiosulphate for 5 minutes.
  16. Wash well with running tap water.
  17. Counterstain with neutral red for 1 minute.
  18. Rinse with tap water.
  19. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Nuclei  –  red
  • Background  –  grey

Notes

  • Ensure that both the ammonium hydroxide and potassium hydroxide are fresh and full strength. Keep both well stoppered when not in use. For the ammonium hydroxide, pour sufficient for use from the stock bottle into a beaker, then immediately restopper the stock bottle. Do not return excess ammonium hydroxide to the stock bottle.
  • After making the ammoniacal silver solution, smell the solution to ensure it has only a faint smell of ammonia. If the smell of ammonia is strong it indicates that too much ammonium hydroxide has been added. If so, it is preferable to make the solution again. Improperly made ammoniacal silver solutions can affect the quality of the impregnation.
  • Drury & Wallington say to bleach with 3% aqueous potassium metabisulphite instead of oxalic acid at step 3.
  • Drury & Wallington also specify treatment with 3% aqueous potassium bisulphite for one minute followed by a distilled water rinse, immediately after toning (step 13). They do not say if this is part of the toning procedure or is an independent step. Culling et. al. omit it.
  • Iron alum is ferric ammonium sulphate.
  • 10% formalin is made by diluting strong formalin 1:10 with tap water (10 mL strong formalin, 90 mL tap water).
  • Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black reticulin fibres on a pale grey-brown background. Others tone longer (a few minutes) to produce black reticulin fibres on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.

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.
    Oxford University Press, London, England.
  2. Culling, C F A, Allison, R T, Barr, W T, (1985).
    Cellular pathology technique., Ed. 4.
    Butterworths, London, England.
March 30, 2023
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Bancroft’s Crystal Violet for Amyloid

By Amyloid, Metachromasia, Protocols, Stain Target, Stain Type
Protocol

Bancroft's Crystal 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 crystal violet solution for 5 minutes.
  3. Rinse with distilled water.
  4. Differentiate briefly with dilute acetic acid for 15 – 20 seconds.
  5. Counterstain with methyl green for 5 – 15 minutes.
  6. Wash with distilled water.
  7. Either drain all water from the slide until just damp then blot and mount with Apathy’s or Highman’s medium, or drain all water from the slide until just damp then blot and flood with xylene. Repeat until the section is cleared, then mount with a resinous medium.

Expected Results

  • Amyloid – purple-red
  • Background – green
  • Nuclei – green

Safety Note

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

References

  1. Bancroft, J. D., (1963).
    Stain technology, v. 38, p. 336.London, England.
March 30, 2023
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Bancroft’s Methyl Green for Amyloid

By Amyloid, Metachromasia, Protocols, Stain Target, Stain Type
Protocol

Bancroft's Methyl Green

for Amyloid

6
steps
2
materials
print

Materials

  • Methyl green, 2% aqueous, washed with chloroform to remove crystal violet
  • Acetic acid, 1% aqueous

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 green solution for 1-5 minutes.
  3. If necessary, differentiate in dilute acetic acid until amyloid is red and contrasts well.
  4. Rinse well with tap water.
  5. Drain all water from the slide until just damp and blot dry.
  6. Flood with triethylphosphate, then with xylene, and coverslip using a resinous medium.

Expected Results

  • Amyloid – purple-red
  • Background – green
  • Nuclei – green

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. 4, p. 222.
    Oxford University Press, London, England.
March 30, 2023
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