Category

Stain Type

Gridley’s Stain for Fungi

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

Gridley's Stain

for Fungi

15
steps
7
materials
print

Materials

  • Schiff’s reagent
  • Aldehyde fuchsin
  • Chromic acid
    MaterialAmount
    Chromium trioxide5g
    Distilled water500mL
  • Bleach
    MaterialAmount
    Sodium metabisulfite5g
    Distilled water500mL
  • Metanil yellow
    MaterialAmount
    Metanil yellow1g
    Distilled water400mL
    Acetic acid, glacial2drops

Tissue Sample

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

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place in chromic acid for 1 hour.
  3. Wash well with tap water.
  4. Treat with the metabisulfite bleach for 1 minute.
  5. Wash well with tap water.
  6. Rinse with distilled water.
  7. Place in Schiff’s reagent for 20 minutes.
  8. Wash well with tap water.
  9. Rinse with 70% ethanol.
  10. Place in aldehyde fuchsin 30 minutes.
  11. Rinse off excess with 95% ethanol.
  12. Wash well with tap water.
  13. Counterstain with metanil yellow for 1 minute.
  14. Rinse well with distilled water.
  15. Dehydrate, clear and mount in a resinous medium.

Expected Results

  • Fungi  –  purple
  • Background  –  yellow

Notes

  • At step 2, a 10% solution of chromic acid applied for 10 minutes will give similar results.

Safety Note

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

References

  1. Bancroft, J.D. and Stevens A. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
  2. Humason, G. L., (1967)
    Animal tissue techniques, Ed. 2
    W. H. Freeman and Company, San Francisco, Ca, USA
March 30, 2023
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Gadsdon’s Alum Hematoxylin

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

Gadsdon's Alum Hematoxylin

8
steps
7
materials
print

This formula is by Prof. Derek Gadsdon at the Sheffield Children’s Hospital, UK.

Materials

MaterialAmountFunction
Hematoxylin5.5 gDye
Potassium alum60 gMordant
Distilled water610 mLSolvent
100% ethanol100 mLSolvent
Glycerol300 mLStabiliser
Sodium iodate0.5 gOxidant
Glacial acetic acid20 mLAcidifier

Compounding procedures

  1. Dissolve the hematoxylin in 100 mL ethanol.
  2. Dissolve the Alum in 600 mL water.
  3. Dissolve the sodium iodate in 10 mL water.
  4. Combine the hematoxylin and Alum solutions. Mix well.
  5. Add the sodium iodate solution. Mix well.
  6. Add the acetic acid. Mix well.
  7. Leave overnight.
  8. Add the glycerol. Mix well.
  9. Filter before use.
  10. The solution may be used immediately.
  11. It is stable at room temperature for at least 6 months.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for the appropriate time.
  3. Rinse well with water.
  4. Differentiate with acid ethanol if using regressively.
  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 solution may be used regressively or progressively.
    • Regressively, staining time is 6 minutes.
    • Progressively, staining time is 3 minutes.
    • Frozen sections will stain in 1 minute.
  • As with many strong formulations mucins may stain 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. Histopathology laboratory, Red Cross Children’s Hospital.
    Gadsdon’s Haematoxylin Method
    Histonet communication, Nov, 1996
  2. Jim Elsam, HTEQA Services.
    Gadsdon’s Hx
    Histonet communication, Nov, 1996
March 30, 2023
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Gage’s Alum Hematoxylin

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

Gage's Alum Hematoxylin

6
steps
5
materials
print

Materials

MaterialAmountFunction
Hematoxylin1 gDye
Potassium alum40 gMordant
Distilled water1 LSolvent
95% ethanol20 mLSolvent
Chloral hydrate20 gOxidant

Protocol

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

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • The formula indicates the solution is progressive.
  • As no oxidising agent is present the solution needs ageing.
  • The appropriate time should be determined by trial, but 5-10 minutes should be adequate.
  • 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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Galigher’s Alum Hematoxylin

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

Galigher's Alum Hematoxylin

8
steps
5
materials
print

This solution was recommended as a substitute for Harris’ Alum hematoxylin.

Materials

MaterialAmountFunction
Hematoxylin5 gDye
Ammonium alum3 gMordant
Distilled water500 mLSolvent
100% ethanol500 mLSolvent
Mercuric oxide6 gOxidant

Compounding procedures

  1. Combine the water and ethanol.
  2. Add the Alum and dye, and bring to a boil.
  3. Add the mercuric oxide, then simmer for 20 minutes.
  4. Restore the volume to 1 L with 50% ethanol.
  5. Cool, and filter through double thickness filter paper.
  6. Store in a tightly stopper bottle.
  7. The solution may be used immediately, and is stable for about six months.

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

  • Mercuric salts are toxic and no longer recommended for oxidation of hematoxylin. Ensure that discarded solution is disposed of as mercury contaminated, or substitute 0.5 grams sodium iodate as the oxidant.
  • Staining capability may continue to improve for a short while.
  • The appropriate 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.
March 30, 2023
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Gallego’s Carbol Fuchsin for Nuclei

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

Gallego's Carbol Fuchsin

for Nuclei

8
steps
3
materials
print

Materials

Solution A

MaterialAmount
Carbol fuchsin2mL
Distilled water98mL

Solution B

MaterialAmount
Formalin, conc. (40%)1mL
Distilled water99mL

Tissue Sample

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

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into solution A for 5 minutes.
  3. Wash with water.
  4. Place into solution B for 5 minutes.
  5. Wash with water.
  6. Stain with a contrast stain.
  7. Dehydrate with ethanol.
  8. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue-black
  • Cytoplasm  –  as counterstain

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:
    Gallego, (1919)
    Trabajos del Laboratorio de investigaciones biológicas de la
    Universidad de Madrid,
    v. 17, pp.95
    And:
    Biot, (1910)
    Compte rendu de l’Association des anatomistes, Congrès de Lyon, pp.234
March 30, 2023
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Garvey’s Alum Hematoxylin

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

Garvey's Alum Hematoxylin

6
steps
6
materials
print

Materials

MaterialAmountFunction
Hematoxylin2.5 gDye
Potassium alum45 gMordant
Distilled water900 mLSolvent
100% ethanol100 mLSolvent
Sodium iodate0.3 gOxidant
Citric acid1 gAcidifier

Compounding procedures

  1. Dissolve the hematoxylin in the ethanol.
  2. Dissolve the Alum in the distilled water with heat.
  3. Combine the two solutions, then add the sodium iodate and citric acid.
  4. Shake well to dissolve.
  5. The solution may be used immediately, and is stable for several months.

Protocol

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

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • Ammonium Alum may be used instead of potassium alum.
  • As the solution was recommended as a substitute for Mayer’s Alum hematoxylin, staining times and results should be comparable. However, the increased hematoxylin content indicates the solution should stain more darkly.
  • 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. Winsome Garvey,
    Modification of the Mayer Hematoxylin stain,
    Journal of Histotechnology, v.14, No.3, p.163
March 30, 2023
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Masson’s HES General Oversight Stain

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

Masson's HES

General Oversight Stain

12
steps
6
materials
print

This is also known as the Hematoxylin Erythrosine Saffron stain.

Materials

  • Regressive hemalum
  • Solution A
    MaterialAmount
    Erythrosine B1g
    Tap water100mL

    Dissolve the erythrosine B and filter. Preserve with a few drops drops of chloroform.

  • Solution B
    MaterialAmount
    Saffron2g
    Distilled water100mL
    Strong formalin1mL
    Tannic acid, 5% aqueous1mL

    Add the saffron stigmata to the water and heat in a boiling water bath for one hour. Filter, and add the formalin and tannic acid. Life is a few weeks.

Tissue Sample

No particular fixative was specified. 5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable but results may be improved by refixing in Bouin’s fixative or saturated aqueous picric acid at 56°C for one hour prior to staining, then washing the sections in tap water to remove all yellow discoloration.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Treat with Bouin’s fluid if desired.
  3. Stain nuclei with hemalum, differentiate and blue.
  4. Wash well with water.
  5. Place in solution A for 2-5 minutes.
  6. Wash rapidly with tap water.
  7. Differentiate erythrosine with 70% ethanol for a few seconds to decolorize collagen.
  8. Wash rapidly with tap water.
  9. Place into solution B for 5 minutes.
  10. Wash rapidly with tap water.
  11. Dehydrate rapidly with absolute ethanol.
  12. Clear with xylene and mount with a synthetic resinous medium.

Expected Results

  • Nuclei  –  blue
  • Cytoplasm  –  red shades
  • Muscle  –  pink
  • Elastic fibres  –  pink
  • Collagen  –  orange-yellow

Notes

  • Saffron is expensive. It may be available in East Indian grocery stores or health food stores as its most common use today is as a spice and food coloring. Usually, whole stigmata are more effective than ground saffron.
  • Eosin B or phloxine B may be substituted for erythrosine B.

Safety Note

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

References

  1. Biological Staining Methods, 6th ed. (1957)
    Gurr, G. T.,
    George T. Gurr, London, UK
  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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HPS Variant General Oversight Stain

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

HPS Variant

General Oversight Stain

10
steps
5
materials
print

This is also known as the Hematoxylin Phloxine Saffron stain.

Materials

  • Regressive hemalum
  • Solution A
    MaterialAmount
    Phloxine B2g
    Distilled water100mL

    Dissolve the phloxine B and filter. Preserve with a crystal of thymol.

  • Solution B
    MaterialAmount
    Saffron30g
    Absolute ethanol1L

    Add the saffron to 500ml absolute ethanol and extract at 60°C for 48 hours. Decant, and store in a dark bottle. Repeat with 500 mL fresh absolute ethanol and combine with the first extraction.

Tissue Sample

No fixative was specified, but most methods using acid dyes benefit from picric acid or mercuric chloride fixation. 5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable but results may be improved by refixing in Bouin’s fixative or saturated aqueous picric acid at 56°C for one hour prior to staining, then washing the sections in tap water to remove all yellow discoloration.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Treat with hemalum for 5 minutes, differentiate and blue.
  3. Wash with running tap water for 5 minutes.
  4. Place in solution A for 5 minutes.
  5. Wash with running tap water for 5 minutes.
  6. Differentiate phloxine with 95% ethanol.
  7. Thoroughly dehydrate with absolute ethanol.
  8. Place into solution B for 5-10 minutes until a suitable red-yellow balance is achieved.
  9. Dehydrate with 4 changes of absolute ethanol.
  10. Clear with 4 changes of xylene and mount with a synthetic resinous medium.

Expected Results

  • Nuclei – blue
  • Cytoplasm – red shades
  • Collagen – yellow

Notes

  • Harris’ hemalum was named, but the formula given was a variant of Bencosme’s hemalum.
  • Saffron is expensive. It may be available in East Indian grocery stores or health food stores as its most common use today is as a spice and food coloring. Usually, whole stigmata are more effective than ground saffron.
  • Store and use the saffron solution at room temperature. It has a limited life of about a month and is best when freshly made. It is important that there be no water in the saffron solution and that sections be thoroughly dehydrated before it is applied. If contaminated with moisture the solution must be discarded.

Safety Note

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

References

  1. Histonet posting
March 30, 2023
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Duval’s Alum Hematoxylin

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

Duval's Alum Hematoxylin

6
steps
4
materials
print

Materials

Original Formula

MaterialAmountFunction
Hematoxylin, concentrated ethanolic8 mLDye
Ammonium or potassium aluma littleMordant
Distilled water800 mLSolvent

Modern Formula

MaterialAmountFunction
Hematoxylin, saturated ethanolic8 mLDye
Ammonium or potassium alum25 gMordant
Distilled water800 mLSolvent

Compounding procedure

  1. Dissolve the alum in the water.
  2. Add the hematoxylin.
  3. The solution is likely progressive, although this is not stated to be so.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for a few 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

  • This solution is from the late 1800’s and is now obsolete, although the modern formula should stain satisfactorily.
  • Concentrated alcoholic hematoxylin, after ripening, would have contained no more than 7% hematein. It was made by soaking logwood chips in ethanol until no more dye dissolved out, then oxidized naturally. Depending on the sample of logwood and the amount of dye it contained, more than one batch may have been necessary to saturate the ethanol.
  • The type of alum was not specified, the most likely being either potassium or ammonium.
  • The formula calls for adding a “little alum” to 800 mL water. Potassium alum saturates at about 14% in water, so 800 mL would contain about 112 g. I have taken just less than 25% of the maximum (i.e. 25 grams) as being a “little”.
    Of course, it could be any amount between 1 and 112 grams.
  • The appropriate time should be determined by trial. The instructions are to use full strength for a few minutes.
  • 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. Arthur Bolles-Lee, (1885)
    The Microtomist’s Vade-Mecum
    Originally published by: J & A Churchill, London, England.
    Republished by: Science Heritage Ltd., Lincolnwood, Illinois, USA.
  2. Susan Budavari, Editor, (1996)
    The Merck Index, Ed. 12
    Merck & Co., Inc., Whitehouse Station, NJ, USA
March 30, 2023
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Ehrlich’s Alum Hematoxylin

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

Ehrlich's Alum Hematoxylin

8
steps
6
materials
print

Materials

MaterialAmountFunction
Hematoxylin6 gDye
95% ethanol300 mLSolvent
Potassium alumexcessMordant
Distilled water300 mLSolvent
Glycerol300 mLStabiliser
Glacial acetic acid30 mLAcidifier

Compounding procedures

  1. Dissolve the hematoxylin in the ethanol mixed with acetic acid.
  2. Dissolve the alum in the water mixed with glycerol in an oversized container.
  3. Add the hematoxylin solution to the alum solution.
  4. Plug the container loosely with cotton wool.
  5. Ripen by leaving in a warm, sunlit place for several weeks.
  6. When sufficiently ripened, store tightly stoppered in a cool, dark place.
  7. The solution is stable for years.

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

  • Gray gives 7 grams hematoxylin, and specifies ammonium alum.
  • The alum should be added to excess. This should be about 50 grams, but enough should be added to ensure undissolved alum is present.
  • This is a strongly staining, regressive formula. The staining time should be determined by trial. Usually, 20 minutes is adequate.
  • As with many strong alum hematoxylin solutions, cartilage, cement lines and mucin may stain blue.
  • The solution may be chemically ripened by adding 0.5g sodium iodate, but chemicallly ripened solutions are inferior in longevity.
  • 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.
    Citing:
    Ehrlich, (1886)
    Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik, v. 3, p. 150.
    Leipzig.
March 30, 2023
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