Category

Stain Type

Gordon & Sweets Impregnation for Reticulin

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

Gordon & Sweets Impregnation

for Reticulin

19
steps
11
materials
print

Materials

  • Silver nitrate, 10% aqu.
  • Strong ammonium hydroxide (s.g. 0.880).
  • Sodium hydroxide, 3% aqu.
  • Oxalic acid, 1% aqu.
  • Iron alum, 2.5% aqu.
  • Formalin, 10% aqu.
  • Yellow gold chloride, 0.2% aqu.
  • Sodium thiosulphate, 3% aqu.
  • Neutral red, 1% aqu.
  • Mallory bleach
    MaterialAmount
    Potassium permanganate, 0.5% aqu.47.5mL
    Sulphuric acid, 3% aqueous2.5mL

Preparation of Ammoniacal Silver

  1. Place 5 mL of the 10% silver nitrate in a 100 mL flask.
  2. Using a pasteur pipette, add a drop of strong ammonium hydroxide then swirl the solution for a few seconds.
  3. A precipitate will form at first.
  4. Continue adding ammonium hydroxide drop by drop and swirling until the precipitate is just redissolved.
  5. Add 5 mL of 3% sodium hydroxide and mix well.
  6. A precipitate will again form.
  7. Add drops of ammonium hydroxide until the precipitate is just redissolved, leaving a faint opalescence.
  8. Dilute to 50 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 the Mallory bleach for 5 minutes.
  3. Bleach in Oxalic acid for a few minutes.
  4. Rinse with distilled water.
  5. Rinse well with tap water.
  6. Sensitise with iron alum solution for 15 minutes.
  7. Rinse well with distilled water.
  8. Treat with ammoniacal silver for 30 seconds.
  9. Rinse well with distilled water.
  10. Reduce in formalin for 1 minute.
  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 sodium thiosulphate for 10 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

  • Gordon & Sweets also suggested a variant that used one of Foot’s ammoniacal silver solutions. Apart from that, the method is the same as given above.

    Foot’s ammoniacal silver

    • To 10 mL of 1% silver nitrate, add 0.1 mL of 40% potassium hydroxide.
    • Add strong ammonium hydroxide by drops until the precipitate just redissolves.
    • Make up the volume to 100mL with distilled water.
  • Ensure that both the ammonium hydroxide and sodium 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.
  • Most references to the Gordon & Sweets’ reticulin stain specify that the ammoniacal silver solution should be made with 10.2% aqueous silver nitrate and 3.1% aqueous sodium hydroxide. No explanation is given. The 10% and 3% solutions respectively, as given by Gray, work satisfactorily.
  • Iron alum is ferric ammonium sulphate. For routine formalin fixed tissue 15 minutes in the iron alum is usually sufficient. If necessary the time may be extended up to 2 hours.
  • 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.
  3. 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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Graham’s Aluminum Hematoxylin

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

Graham's Aluminum Hematoxylin

6
steps
5
materials
print

Grahams Alum hematoxylin is a modification of Gill’s Alum hematoxylin, and was designed for routine staining of plant tissue. It should also be satisfactory for animal tissues. It has been formulated so that small quantities may be prepared fresh and sufficient for a single Coplin jar.

Materials

MaterialAmountFunction
Distilled water750 mLSolvent
Propylene glycol210 mLSolvent
Glacial acetic acid20 mLAcidifier
Aluminum sulphate17.6 gMordant
Sodium iodate0.2 gOxidant
MaterialAmountFunction
Solution BAmountFunction
Hematoxylin10 gDye
Propylene glycol100 mLSolvent

Compounding Procedures

Solution A

  1. Combine all the ingredients and filter when dissolved.

Solution B

  1. Shake vigorously several times during 48 hours until the hematoxylin dissolves.

Working solution

  1. Solution A – 49 mL
  2. Solution B – 1 mL
  3. Mix well.
  4. It is ready for use when an opaque dark purple.

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

  • Staining is progressive, application time being determined by trial (try 2 minutes).
  • Propylene glycol replaces toxic ethylene glycol.
  • 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.
  • The aluminum sulphate should be Al2(SO4)3.18H2O. Adjust the amount if different.

Safety Note

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

References

  1. Graham, E. T., (1991)
    A quick mix aluminum hematoxylin stain
    Biotechnic and Histochemistry, pp. 279-281
March 30, 2023
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Hamilton Alum Hematoxylin

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

Hamilton Alum Hematoxylin

6
steps
6
materials
print

This formula is based on Langeron’s 1942 (Mayer’s) formula. It has been referred to as Hamilton Alum hematoxylin as it was received via the internet from a histotechnologist at the Hamilton Health Sciences Corporation, Hamilton, Ontario, Canada.

Materials

MaterialAmountFunction
Hematoxylin2 gDye
Ammonium alum75 gMordant
Distilled water950 mLSolvent
100% ethanol50 mLSolvent
Sodium iodate0.3 gOxidant
Glacial acetic acid30 mLAcidifier

Compounding Procedure

  1. Dissolve the Alum in the water.
  2. Add the ethanol and mix well.
  3. Add the hematoxylin and dissolve.
  4. Add the sodium iodate and mix well.
  5. Leave overnight in the dark to ripen.
  6. Mix well, and add the acetic acid.
  7. 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 3 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

  • Potassium Alum may be used instead of ammonium alum.
  • The solution is progressive.
  • 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

Personal internet communication, source unidentified.

March 30, 2023
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Hansen’s Iron Hematoxylin

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

Hansen's Iron Hematoxylin

8
steps
3
materials
print

Materials

MaterialAmountFunction
Hematoxylin0.75 gDye
Ferric ammonium sulfate4.5 gMordant
Distilled water100 mLSolvent

Compounding Procedure

  1. Dissolve the iron alum in 65 mL of the water.
  2. Dissolve the hematoxylin in the remaining water.
  3. Combine the solutions and bring to a boil.
  4. Cool and filter.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse with tap water.
  4. Treat 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 stability of the solution is not given, but the simplicity of the formula indicates it would not be stable for too long.
  • Although a staining time was not given, 30 minutes should be sufficient. Determine 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:
    Hansen, (1905)
    Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik,
    v.22, p.55. Leipzig.
March 30, 2023
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Harris’ Alum Hematoxylin

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

Harris' Alum Hematoxylin

8
steps
6
materials
print

Materials

MaterialAmountFunction
Hematoxylin5 gDye
Potassium alum100 gMordant
Distilled water1 LSolvent
95% ethanol50 mLSolvent
Mercuric oxide2.5 gOxidant
Glacial acetic acid40 mLAcidifier

Compounding Procedure

  1. Dissolve the hematoxylin in the ethanol.
  2. Dissolve the Alum in warmed distilled water.
  3. Combine the solutions in an oversize container.
  4. Bring to a boil.
  5. Add the mercuric oxide, remove from heat.
  6. Cool rapidly.
  7. Add the glacial acetic acid.
  8. It may be used immediately when cool.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Paraffin sections should be stained for 5 minutes, and cytology smears for 45 seconds.
  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 solution is a regressive formulation, although it is sometimes used progressively with short staining times.
  • When the mercuric oxide is added, the solution may foam.
  • Mercuric oxide is toxic and no longer recommended for oxidation of hematoxylin. If used, all discarded solution should be disposed of as mercury contaminated. Sodium iodate is sometimes substituted, based on a maximum of 0.2 gram sodium iodate for each 1 gram of hematoxylin. In practice, 0.5 gram works quite well.
  • The acetic acid is optional, but nuclear staining is more precise if it is included.
  • A precipitate often forms, so the solution should be filtered before use.
  • As with many strong hematoxylin solutions, mucin may be blue.
  • 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.
March 30, 2023
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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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