Category

Protocols

[facetwp template="protocol_by_facet"]

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
Love0

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
Love0

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
Love0

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
Love0

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
Love0

French’s Stain for Elastic Fibres

By Dye Type, Elastic Fibers, Iron Resorcin, Protocols, Stain Target, Stain Type
Protocol

French's Stain

for Elastic Fibres

7
steps
9
materials
print

Materials

  • Weigert’s iron hematoxylin or equivalent
  • Van Gieson’s picro-fuchsin
  • French’s solution
    MaterialAmount
    Basic fuchsin1g
    Crystal violet1g
    Resorcin4g
    Ferric chloride, 30% aqu.25mL
    Ethanol 95%200mL
    Distilled water200mL
    Hydrochloric acid, conc.4mL

    Preparation

    1. Add the dye and resorcin to the water in an oversize flask.
    2. Bring to the boil, and add the ferric chloride.
    3. Boil for 5 minutes.
    4. Cool and filter.
    5. Dry the filter paper and beaker. Place the precipitate and filter paper back into the flask.
    6. Add the ethanol and heat carefully until the precipitate dissolves.
    7. Add the hydrochloric acid.
    8. Restore to 200 mL with 95% ethanol.

Tissue Sample

5µ 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 French’s solution until adequately stained (may be overnight).
  3. Wash with 95% ethanol to remove excess solution.
  4. Differentiate with 1% acid alcohol.
  5. Wash in water.
  6. Counterstain with iron hematoxylin and van Gieson.
  7. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  dark blue-green
  • Collagen  –  red
  • Cytoplasm  –  yellow
  • Nuclei  –  blue

Notes

  • This is Weigert’s iron resorcin fuchsin using crystal violet and basic fuchsin instead of basic fuchsin alone.
  • It is usually recommended that the basic fuchsin should not be of the type that produces good Schiff’s reagent. That is, the basic fuchsin should contain more rosanilin than pararosanilin.
  • The ferric chloride solution should be freshly made.

Safety Note

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

References

  1. Histological demonstration techniques, (1974)
    Cook, H C.
    Butterworths, London, England
March 30, 2023
Love0

Fullmer & Lillie’s Orcinol-New Fuchsin for Elastic Fibres

By Dye Type, Elastic Fibers, Iron Resorcin, Protocols, Stain Target, Stain Type
Protocol

Fullmer & Lillie's Orcinol-New Fuchsin

for Elastic Fibres

5
steps
7
materials
print

Materials

    • Weigert’s iron hematoxylin or equivalent
    • Van Gieson’s picro-fuchsin
    • Orcinol-New Fuchsin solution
      MaterialAmount
      New fuchsin2g
      Orcinol4g
      Ferric chloride, 30% aqu.50mL
      Distilled water200mL
      Ethanol 95%100mL

      Preparation

      1. Add the dye and orcinol to the water in an oversize flask.
      2. Boil for 5 minutes.
      3. Add the ferric chloride.
      4. Boil for a further 5 minutes.
      5. Cool and filter.
      6. Dry the filter paper and beaker. Place the precipitate and filter paper back into the flask.
      7. Add the ethanol and heat carefully until the precipitate dissolves.
      8. Restore to 100 mL with 95% ethanol.

Tissue Sample

5µ 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 orcinol-new fuchsin solution for 15 minutes at 37°C.
  3. Wash with 3 changes of 70% ethanol for 5 minutes each.
  4. Counterstain with iron hematoxylin and van Gieson.
  5. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  deep violet
  • Collagen  –  red
  • Cytoplasm  –  yellow
  • Nuclei  –  black

Notes

  • Fullmer and Lillie specified 25mL USP IX Liquor Ferri Chloridi, or a solution of 15.5g ferric chloride (FeCl3.6H2O) made up to 25 mL with water, i.e. 25mL of a 62% solution. In either case, it should be fresh.

Safety Note

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

References

  1. Fullmer, H M and Lillie, R D, (1956).
    A selective stain for elastic tissue (orcinol-new fuchsin).,
    Stain Technologyl, v 31, page 27-29.
  2. Lillie, R D and Fullmer, H M, (1976).
    Histopathological technic and practical histochemistry, Ed. 4. pp. 714.
    McGraw-Hill, New York, USA.
March 30, 2023
Love0

Hart’s Stain for Elastic Fibres

By Dye Type, Elastic Fibers, Iron Resorcin, Protocols, Stain Target, Stain Type
Protocol

Hart's Stain

for Elastic Fibres

7
steps
4
materials
print

Materials

Tissue Sample

5µ 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 Hart’s solution overnight.
  3. Wash with 95% ethanol to remove excess solution.
  4. Differentiate with 1% acid alcohol.
  5. Wash with tap water.
  6. Counterstain with iron hematoxylin and van Gieson.
  7. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  blue black
  • Collagen  –  red
  • Cytoplasm  –  yellow
  • Nuclei  –  blue

Notes

  • This modification should be used if poor staining is obtained using Weigert’s method.
  • The optimum dilution may be more or less than given.

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., (1974)
    Handbook of histopathological and histochemical techniques Ed. 3
    Butterworth, London, UK.
March 30, 2023
Love0

Humberstone’s Stain for Elastic Fibres

By Dye Type, Elastic Fibers, Iron Resorcin, Protocols, Stain Target, Stain Type
Protocol

Humberstone's Stain

for Elastic Fibres

8
steps
12
materials
print

Materials

  • Weigert’s iron hematoxylin or equivalent
  • Van Gieson’s picro-fuchsin
  • Potassium permanganate, 0.25% aqu., for the Mallory bleach
  • Humberstone’s solution
    MaterialAmount
    Victoria blue 4R1g
    Ethyl violet1g
    Resorcin4g
    Dextrin0.5g
    Ferric chloride, 30% aqu.25mL
    Ethanol, 95%150mL
    Phenol10g
    Hydrochloric acid, conc.4mL
    Distilled water200mL

      • Preparation

      1. In an oversized flask, add the dyes to the water, and boil to dissolve.
      2. Add the resorcin, dextrin and ferric chloride. Boil for 3 minutes. Cool and filter.
      3. Place the precipitate and filter paper back into the original flask. Add 100 ml of 95% ethanol and ently for 15 minutes.
      4. Cool and filter.
      5. Make up the volume to 350 mL with 95% ethanol.
      6. Add the phenol and hydrochloric acid.

Tissue Sample

5µ 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. Do a Mallory bleach with 0.25% potassium permanganate for ten minutes.
  3. Rinse with 95% ethanol.
  4. Place into Humberstone’s solution overnight.
  5. Wash with 95% ethanol to remove excess solution.
  6. Wash in water.
  7. Counterstain with iron hematoxylin and van Gieson.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  blue-black
  • Nuclei  –  black
  • Cytoplasm  –  yellow
  • Collagen  –  red

Notes

  • The authors state that buffered formalin is an unsuitable fixative. I have not found this so and have used this method regularly with such tissue. It may be that the modern practice of minimalist fixation limits the effect.
  • As the solution ages, the demonstration of elastic fibres improves in both quality and speed. When the solution is a few years old elastic is demonstrated within about 4 hours, although overnight is generally
    preferable.
  • The solution is stable for more than ten years. Due to the improvement in staining as the solution ages, it is suggested that a litre, or more, be made at a time.
  • The authors suggest as suitable counterstains, iron hematoxylin and van Gieson, Masson’s trichrome or MSB (with green collagen).

Safety Note

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

References

  1. Humberstone, G. C. W., and Humberstone, F. D., (1969)
    An Elastic Tissue Stain
    Journal of Medical Laboratory Technology, V. 26, No 2, pp. 99.
March 30, 2023
Love0

Miller’s Stain for Elastic Fibres

By Dye Type, Elastic Fibers, Iron Resorcin, Protocols, Stain Target, Stain Type
Protocol

Miller's Stain

for Elastic Fibres

8
steps
12
materials
print

Materials

Preparation

  1. In an oversized flask heat the water and add the three dyes.
  2. When dissolved, add the resorcin, dextrin and ferric chloride in the order given.
  3. Boil for 5 minutes, then filter while hot.
  4. Place the precipitate and filter paper back into the original flask, add the 95% ethanol and simmer gently for 20 minutes.
  5. Cool and filter.
  6. Restore the volume to 200 mL with 95% ethanol, then add 2 mL of concentrated hydrochloric acid.

Tissue Sample

5µ 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. Do a Mallory bleach with 0.25% potassium permanganate for ten minutes.
  3. Rinse with 95% ethanol.
  4. Place into Miller’s solution up to three hours.
  5. Wash with 95% ethanol to remove excess solution.
  6. Wash in water.
  7. Counterstain with iron hematoxylin and van Gieson.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

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

Notes

  • Three hours is recommended, although staining may be adequate after 90 minutes.
  • Dilute with an equal quantity of 95% ethanol for overnight staining.
  • The staining solution is stable for years.

Safety Note

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

References

  1. Miller, P.J., (1971)
    Journal of Medical Laboratory Technology, V. 28
  2. Ellis, R.,
    Miller’s Elastic Staining Protocol
    IHC World
March 30, 2023
Love0