Category

Stain Type

McLachlan’s Alum Hematoxylin

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

McLachlan's Alum Hematoxylin

6
steps
7
materials
print

This formulation has been named in honor of Dr. H.K.I. McLachlan FRCPA at the request of the originator, Mike Rentsch.

Materials

MaterialVariantFunction
Var IVar II
Hematoxylin2 g2 gDye
Aluminum sulphate17.5 g–Mordant
Ammonium alum–25 gMordant
Distilled water700 mL700 mLSolvent
Glycerol300 mL300 mLStabiliser
Sodium iodate0.2 g0.2 gOxidant
Glacial acetic acid20 mL20 mLAcidifier

Compounding Procedure

  1. Dissolve the aluminum sulphate or the Alum in about 500 mL water.
  2. Add the hematoxylin and dissolve.
  3. Add the acetic acid and mix well.
  4. Warm the glycerol to reduce viscosity, and add to the mixture.
  5. Mix well, then make up to 1 litre with water.
  6. Add the iodate and mix for 5-10 minutes.
  7. Store in a tightly capped bottle in the dark.
  8. The solution remains usable for 2-5 years.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place paraffin sections in the staining solution for 2-4 minutes and frozen sections for 40-60 seconds.
  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 is a progressive solution.
  • 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. Rentsch, M.
    Australian Biostain P/L.
    Personal communication via the internet.
March 30, 2023
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Horneyold’s Alum Hematoxylin

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

Horneyold's Alum Hematoxylin

8
steps
6
materials
print

Materials

Hematoxylin Solution

MaterialAmountFunction
Hematoxylin0.7 gDye
100% ethanol20 mLSolvent

Alum Solution

MaterialAmountFunction
Ammonium alum0.35 gMordant
Distilled water60 mLSolvent

Tincture of Iodine

MaterialAmountFunction
Iodine70 gOxidant
Potassium iodide50 g–
Distilled water50 mLSolvent
100% ethanol950 mLSolvent

Compounding Procedures

Tincture of iodine

  1. Mix the dry iodine and potassium iodide together.
  2. Add the water and mix well to dissolve.
  3. Add the ethanol and mix well.

Staining solution

  1. Dissolve the hematoxylin in the ethanol and the Alum in the water.
  2. Combine the two solutions and mix well.
  3. Leave in the light by a window for 3-4 days.
  4. Add 20 drops tincture of iodine.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 5-10 minutes.
  3. Rinse well with water.
  4. Differentiate with acetic ethanol.
  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

  • Horneyold’s formula is a modification of Böhmer’s Alum hematoxylin.
  • This solution is regressive, and is recommended for osmium fixed tissue.
  • Acetic ethanol is 70% ethanol containing glacial acetic acid. The amount of acetic acid to add was not given, but 0.5%-1% should suffice.
  • 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.
  2. Lange, N.A., (1966)
    Lange’s Handbook of Chemistry, Revised 10th ed.,
    McGraw-Hill.
March 30, 2023
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Lendrum’s Phloxine Tartrazine for Viral Inclusion Bodies

By Intracytoplasmic Granules, Paneth Cells, Protocols, Stain Target, Stain Type, Trichrome Staining, Yellowsolve Staining
Protocol

Lendrum's Phloxine Tartrazine

for Viral Inclusion Bodies

11
steps
6
materials
print

This method is also used for the demonstration of Paneth cell granules, and may be used as a substitute for the HPS if the differentiation in the tartrazine solution is shortened to retain pink cytoplasm and muscle.

Materials

  • Mayer’s hemalum
  • Solution A
    MaterialAmount
    Phloxine B0.5g
    Calcium chloride0.5g
    Distilled water100mL
  • Solution B
    MaterialAmount
    Tartrazineto saturation
    2-Ethoxy ethanol (cellosolve)100mL

Tissue Sample

5µ paraffin sections of formal sublimate fixed tissue is preferred. Formalin fixed tissue is suitable. Other fixatives are likely to be satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei to medium density with hemalum.
  3. Wash in tap water for 5 minutes. Blueing takes place in the phloxine solution.
  4. Place in solution A for 20 minutes.
  5. Rinse in tap water, blot almost dry. Some technologists rinse with cellosolve instead of blotting. The object is to remove all traces of water, as it interferes with the ability of tartrazine to extract phloxine and counterstain.
  6. Rinse with solution B to remove remaining water. Discard solution.
  7. Place in solution B until inclusions are red and all other tissue is yellow. The time varies considerably. Control microscopically.
  8. Rinse thoroughly but briefly with absolute ethanol.
    Do not rinse with water at this stage as it rapidly removes the tartrazine. Some technologists use cellosolve instead of ethanol.
  9. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Acidophil virus inclusion bodies  –  red
  • Paneth cell granules  –  red
  • Background  –  yellow

Notes

  • The 2-ethoxy ethanol must not be replaced by any other solvent.
  • The 2-ethoxy ethanol must be, and must remain, completely anhydrous.

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.
March 30, 2023
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Jalowy’s Impregnation for Reticulin

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

Jalowy's Impregnation

for Reticulin

8
steps
4
materials
print

Materials

  • Silver nitrate, 10% aqu.
  • Strong ammonium hydroxide (s.g. 0.88).
  • Ammonium hydroxide, 1% aqu.
  • Formalin, 10% aqu.

Preparation of Jalowy’s Ammoniacal Silver

  1. Place 20 mL of 10% silver nitrate in a flask and add 1 mL of 40% sodium hydroxide.
  2. Mix well, then filter. Wash the precipitate well with distilled water several times and decant.
  3. Add 20 mL distilled water to the precipitate and suspend.
  4. Add strong ammonium hydroxide by drops until the precipitate is just dissolved.
  5. Dilute to 100 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. Rinse with distilled water.
  3. Place in prewarmed ammoniacal silver solution for 5-30 minutes at 30°C.
  4. Rinse with distilled water.
  5. Rinse with 1% ammonium hydroxide.
  6. Place in 10% formalin for 2-10 minutes.
  7. Rinse well with tap water.
  8. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Background  –  brown

Notes

  • This method was recommended for collagen and reticulin fibres on formalin fixed skin.
  • Ensure that the ammonium hydroxide and sodium hydroxide are fresh and full strength. Keep the ammonium hydroxide well stoppered when not in use. Pour sufficient for use into a beaker, then immediately restopper the stock container. Do not return unused ammonium hydroxide to the stock bottle.
  • The instructions for this method do not specify to tone, fix, or counterstain. If fading or a lack of contrast make it desirable, toning with 0.1% yellow gold chloride until satisfactory, and/or fixing with 5% sodium thiosulphate, and/or counterstaining with 1% aqueous neutral red may be used.

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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Janssen’s Iron Hematoxylin

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

Janssen's Iron Hematoxylin

8
steps
6
materials
print

Materials

MaterialAmountFunction
Hematoxylin1 gDye
Ferric ammonium sulfate5 gMordant
100% ethanol5 mLSolvent
Distilled water70 mLSolvent
Glycerol15 mLSolvent
Methanol15 mLSolvent

Compounding Procedure

Gray Method

  1. Dissolve the iron alum into the water.
  2. Dissolve the hematoxylin into the ethanol.
  3. Combine, and leave for one week at room temperature.
  4. Filter, and then add the other ingredients.

Lillie Method

  1. Combines all solutions immediately once they are dissolved, and omits the week at room temperature.
  2. The solution is stable for a few months.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 15-30 minutes.
  3. Rinse with tap water.
  4. 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

  • This solution may be used as an acid resistant nuclear stain.
  • It is recommended as a substitute for Weigert’s iron hematoxylin.

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:
    Janssen, (1897),
    Cellule, v.14, p.207.
    Lillie & Earle, (1939),
    Stain Technology, v.14, p.53
March 30, 2023
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Kefalas’s Iron Hematoxylin

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

Kefalas's Iron Hematoxylin

16
steps
4
materials
print

Materials

Solution A

MaterialAmountFunction
Hematoxylin1 gDye
Ferric chloride1 gMordant
Acetone100 mLSolvent
Hydrochloric acid0.05 mLAcidifier

Compounding Procedure

  1. Dissolve the ferric chloride and hematoxylin in the acetone.
  2. Add the hydrochloric acid.

Protocol

Standard Method

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 30 minutes.
  3. Wash well in running tap water to blue.
  4. Rinse with distilled water.
  5. Counterstain if desired.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Alternative Method

Since this solution is almost anhydrous it can be used in a staining procedure which completely avoids water or ethanol.

  1. Remove wax with xylene.
  2. Remove xylene with a few changes of acetone.
  3. Place into staining solution for 30 minutes.
  4. Wash with acetone.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Background  –  as counterstain or unstained

Notes

  • 0.05 mL hydrochloric acid would be about 1-2 drops.
  • The simplicity of the formula indicates it is likely not stable for long.
  • The presence of hydrochloric acid may indicate progressive staining.
  • It is likely suitable as an acid resistant nuclear stain.
  • Although the staining time should be established by trial and error, 30 minutes would likely suffice as a starting point.

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:
    Kefalas, (1926),
    Journal of the Royal Microscopical Society
    v. 46, p. 277.
March 30, 2023
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Kiernan’s Eriochrome Cyanin for Nuclei

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

Kiernan's Eriochrome Cyanin

for Nuclei

8
steps
6
materials
print

This is a substitute for alum hematoxylin in the H & E stain.

Materials

Solution A

MaterialAmount
Eriochrome cyanine R1.0g
Ferric chloride, 5.6%20mL
Sulphuric acid, conc.2.5mL
Distilled waterup to 500 mL

Solution B

MaterialAmount
Hydrochloric acid, conc.5mL
Ethanol, abs.500mL
Distilled water500mL

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 sections into solution A for 5 minutes.
  3. Wash with running tap water for about 30 seconds.
  4. Differentiate with solution B until nuclei are sharply stained, about 10-30 seconds.
  5. Wash with running tap water for about 5 minutes.
  6. Counterstain with eosin or another contrasting dye, as wished.
  7. Dehydrate with ethanol.
  8. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstained

Notes

  • Solution A is stable for years.
  • Solution A is also used for Kiernan’s myelin staining variant.
  • Kiernan also describes a two colour staining variant.
  • Eriochrome cyanine R is also known as mordant blue 3 and solochrome cyanine R.

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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Kiernan’s Eriochrome Cyanin with two colours

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

Kiernan's Eriochrome Cyanin

with two colours

5
steps
4
materials
print

Materials

Solution A

MaterialAmount
Eriochrome cyanine R1.0g
Ferric chloride, 5.6%2.5mL
Sulphuric acid, conc.2.5mL
Distilled waterup to 500mL

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 sections into solution A for 3 minutes.
  3. Rinse with distilled water three times for about 20 seconds each time.
  4. Dehydrate with ethanols.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  purple blue
  • Background  –  red

Notes

  • Solution A is stable for years.
  • Solution A must have a pH of 1.5 for the two colour effect. Check periodically, and adjust with 1M sulphuric or hydrochloric acids, or sodium hydroxide.
  • Kiernan’s also describes regressive nuclear staining and myelin staining variants.
  • Eriochrome cyanine R is also known as mordant blue 3 and solochrome cyanine R

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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Klatskin’s Modification of Masson’s Trichrome for Liver

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

Klatskin's Modification of Masson's Trichrome

for Liver

12
steps
8
materials
print

Materials

  • Harris’ alum hematoxylin.
  • Solution A
    MaterialAmount
    Picric acidto saturation
    Ethanol, 95%100mL
  • Solution B
    MaterialAmount
    Xylidine ponceau1g
    Distilled water99mL
    Glacial acetic acid1mL
  • Solution C
    MaterialAmount
    Phosphomolybdic acid1g
    Distilled water100mL
  • Solution D
    MaterialAmount
    Aniline blueto saturation
    Glacial acetic acid2.5mL
    Distilled water97.5mL
  • Solution E
    MaterialAmount
    Acetic acid, glacial1mL
    Distilled water99mL

Tissue Sample

3-5 micron paraffin sections of formalin fixed liver.

Protocol

  1. Bring sections to water via xylene and ethanol
  2. Stain with Harris’ hemalum for 10 minutes.
  3. Rinse with 95% ethanol.
  4. Place in solution A for 10 minutes.
  5. Wash with water for 10 minutes.
  6. Place in solution B for 5 minutes.
  7. Place in solution C for 5 minutes.
  8. Place in solution D for 2 minutes.
  9. Replace in solution C for 5 minutes.
  10. Place in solution E for 5 minutes.
  11. Place in 70% ethanol for 2 minutes.
  12. Dehydrate with ethanol, clear and mount with a resinous medium.

Expected Results

  • Cytoplasm  –  red
  • Erythrocytes  –  red
  • Muscle  –  red
  • Collagen  –  blue
  • Nuclei  –   brown

Notes

  • Aniline blue, is a mixture of two dyes, methyl blue, and water blue. Either may usually be substituted satisfactorily.
  • Although recommended for liver sections, this modification may also be used as a general replaceement for Masson’s method.

Safety Note

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

References

  1. Petersen, K.F., West, A.B., Reuben, A., Rothman, D. and Shulman, G.I. (1996)
    Noninvasive Assessment of Hepatic Triglyceride Content in Humans With 13C Nuclear Magnetic Resonance Spectroscopy.
    Hepatology, V. 24, p 114-117
March 30, 2023
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Koneff’s Trichrome for Pituitary Cells

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

Koneff's Trichrome

for Pituitary Cells

17
steps
11
materials
print

Materials

Solution A

MaterialAmount
Aniline0.1mL
Ethanol, 90%100mL

Solution B

MaterialAmount
Acetic acid, glacial1mL
Ethanol, 90%100mL

Solution C

MaterialAmount
Azocarmine1g
Acetic acid, glacial1g
Distilled water100mL

Solution D

MaterialAmount
Aniline0.06mL
Ethanol, 90%100mL

Solution E

MaterialAmount
Phosphotungstic acid5g
Distilled water100mL

Solution F

MaterialAmount
Aniline blue0.5g
Orange G2g
Oxalic acid2g
Phosphotungstic acid0.05g
Distilled water100mL

Solution G

MaterialAmount
Acetic acid1mL
Distilled water100mL

Tissue Sample

The instructions specify fixation in see Baley’s fixative. Other mercuric-chrome fixatives would probably be satisfactory. The instructions also specify 3-4 µ celloidin sections which have been attached to a slide, and from which the celloidin has been removed. Possibly, similar paraffin sections would be satisfactory.

Baley’s fixative consists of:

MaterialAmount
Mercuric chloride3.4g
Potassium dichromate3.4g
Concentrated formalin (40%)25mL
Water225mL
Return to fixation instructions.

Protocol

  1. Attach sections to slides and remove celloidin, or dewax sections with xylene and bring to ethanol.
  2. Although not specified, a water rinse followed by the iodine thiosulphate sequence and a second water wash, to remove mercury pigment would be advantageous at this point.
  3. Wash with 70% ethanol.
  4. Place into solution A for 45 minutes.
  5. Place into solution B for 1-2 minutes.
  6. Place into solution C for 2 hours at 56°C.
  7. Wash with water.
  8. Place into solution D until nuclei are red and the cytoplasm is pink.
  9. Place into solution B for 1-2 minutes.
  10. Place into solution E for 4 hours
  11. Place into solution F for 4 hours until basophils are blue.
  12. Return to solution E for 3-5 minutes.
  13. Wash with water.
  14. Rinse with solution G.
  15. Wash with water.
  16. Dehydrate with absolute ethanol.
  17. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  red
  • Acidophils  –  orange-red
  • Basophils  –  blue
  • Chromophobes  –  pale grey

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:
    Koneff, (1938)
    Stain Technology, v.13, pp.49
March 30, 2023
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