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Hematoxylin Alternatives

Nuclear Fast Red for Nuclei

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

Nuclear Fast Red

for Nuclei

7
steps
5
materials

Materials

Solution A

MaterialAmount
Nuclear fast red1g
Potassium aluminum sulphate50g
Distilled water500mL

Preparation

  1. Dissolve the dye and alum into the water.
  2. Boil for 5 minutes. Cool and filter.

Solution B

MaterialAmount
Tartrazine1g
Distilled water1L

Tissue Sample

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

Protocol

  1. Perform the main staining method.
  2. Place into solution A for 5 to 10 minutes
  3. Rinse with water.
  4. Optionally, place into solution B for 30 seconds.
  5. Rinse with distilled water.
  6. Return to the main staining method, or
  7. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  red
  • Cytoplasm  –  yellow or unstained
  • Other tissues  –  according to the main staining method

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.

Neutral Red for Nuclei

By Dye Type, Hematoxylin Alternatives, Protocols, Stain Type

Neutral Red

for Nuclei

4
steps
3
materials

Materials

Staining Solution

MaterialAmount
Neutral red1g
Acetic acid, glacial1drop
Distilled water100mL

To avoid clumping, dust the dye onto the surface of the water while shaking it. Leave overnight, add the acid, and filter.

Tissue Sample

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

Protocol

  1. Complete the primary stain.
  2. Place into the solution for 1 – 3 minutes.
  3. Rinse well with water.
  4. Dehydrate with ethanol and clear with xylene.

Expected Results

  • Nuclei  –  red
  • Other tissues  –  as the primary stain

Notes

  • The small amount of acetic acid slightly acidifies the solution and reduces background staining, making the solution more selectively nuclear. Do not add more than specified.
  • The ethanol used for dehydration removes some of the stain. This permits a small degree of control.
  • If necessary, staining can be removed with acid alcohol. However, check that the primary stain will not be affected before doing so.

Safety Note

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

Lendrum & Mcfarlane Iron Alum-Celestine Blue for Nuclei

By Dye Type, Hematoxylin Alternatives, Protocols, Stain Type

Lendrum & Mcfarlane Iron Alum-Celestine Blue

for Nuclei

6
steps
5
materials

Materials

  • An eosin solution, or other counterstain
  • Iron alum-celestine blue
    MaterialAmount
    Ferric ammonium sulfate5g
    Celestine blue B0.5g
    Distilled water100mL
    Glycerol14mL

Preparation

  1. Dissolve the ferric ammonium sulfate in the distilled water.
  2. Add the celestine blue B.
  3. Boil for 5 minutes.
  4. Cool and filter.
  5. Add the glycerol.

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 the iron alum-celestine blue solution for 3 minutes to 2 hours.
  3. Wash with tap water.
  4. Counterstain with eosin or other counterstain.
  5. Dehydrate with 95% and absolute ethanols.
  6. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Cytoplasm  –  pink

Notes

  • The iron alum-celestine blue solution is stable for a few months.
  • A staining time of 5-10 minutes is usually satisfactory for formalin fixed tissues.
  • This method has been recommended as a substitute for Hematoxylin and Eosin.
  • The celestine blue solution is a modification of that recommended by Proescher and Arkush, and contains added glycerol.
  • Gallamine blue and gallocyanin have also been recommended in this method. Neither dye is as stable as celestine blue, although the colour with gallocyanin most closely resembles alum hematoxylin of all three dyes.

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:
    Lendrum and McFarlane, (1940)
    Journal of Pathology and Bacteriology, v. 50, pp. 381

Llewellyn’s Mordant Blue 3 for Nuclei

By Dye Type, Hematoxylin Alternatives, Protocols, Stain Type

Llewellyn's Mordant Blue 3

for Nuclei

9
steps
8
materials

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

Materials

  • An aqueous or alcoholic eosin Y solution
  • Solution A
    MaterialVariant
    19741978a1978b
    Mordant blue 30.25g1g1g
    Iron alum, 10% aqu.40mL90mL5mL
    Sulphuric acid5mL
    Hydrochloric acid10mL
    Acetic acid50mL
    Distilled water955mL860mL985mL

    For all variants:

    1. Dilute the iron alum with the water.
    2. Add the mordant blue 3 and dissolve.
    3. Add the specified acid, mix and filter.

    The solutions are usable immediately and they have a shelf life of more than one year.

  • Solution B
    MaterialVariant
    Var AVar B
    Hydrochloric acid, conc1mL1mL
    Ethanol, 70%99mL
    Distilled water99mL
  • Solution C
    MaterialAmount
    Sodium acetate0.5g
    Distilled water100mL

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. Rinse well with tap water.
  4. Differentiate with a variant of solution B:
    1. For the 1974 formula: var A for 1-3 seconds or var B for 30 seconds.
    2. For the 1978a formula: var A for about ten seconds.
    3. For the 1978b formula: do not differentiate. Proceed to step 6.
  5. Rinse well with tap water.
  6. Blue with solution C for about 30 seconds.
  7. Rinse well with tap water.
  8. Counterstain with an eosin solution.
  9. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Cytoplasm  –  shades of pink

Human kidney stained progressively with mordant blue 3 and eosin Y at 40x magnification

Human kidney stained progressively with mordant blue 3 and eosin Y at 40x magnification

Human kidney stained progressively with mordant blue 3 and eosin Y at 100x magnification

Human kidney stained progressively with mordant blue 3 and eosin Y at 100x magnification

Human kidney stained progressively with mordant blue 3 and eosin Y at 400x magnification

Human kidney stained progressively with mordant blue 3 and eosin Y at 400x magnification

Notes

  • At times hematoxylin powder has become difficult, if not impossible, to buy. Due to the importance of this dye in making alum hematoxylin for H&E stains, several other dyes were investigated as substitutes. Mordant blue 3 was one of these, and Llewellyn published three variants of this substitute, both progressive and regressive.
  • The 1974 and 1978a formulae are regressive. The 1978b formula is progressive.
  • The sections will be blue after solution A. The blueing referred to in step 6 will deepen this, but it’s main purpose is to remove any pink staining due to unmordanted dye.
  • Iron alum is ferric ammonium sulphate:  FeNH4(SO4)2·12H2O. Use clean crystals.

Safety Note

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

References

  1. Llewellyn, B. D., 1974
    Mordant blue3: A readily available substitute for hematoxylin
    in the routine hematoxylin and eosin stain.
    Stain Technology, v. 49, pp. 347.
  2. Llewellyn, B. D., 1978
    Improved nuclear staining with mordant blue 3 as a hematoxylin substitute
    Stain Technology, v. 53, pp. 73.

Kiernan’s Eriochrome Cyanin for Nuclei

By Dye Type, Hematoxylin Alternatives, Protocols, Stain Type

Kiernan's Eriochrome Cyanin

for Nuclei

8
steps
6
materials

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.

Kiernan’s Eriochrome Cyanin with two colours

By Dye Type, Hematoxylin Alternatives, Protocols, Stain Type

Kiernan's Eriochrome Cyanin

with two colours

5
steps
4
materials

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.

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

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

Duprès’ Magenta for Nuclei

By Dye Type, Hematoxylin Alternatives, Protocols, Stain Type

Duprès' Magenta

for Nuclei

7
steps
6
materials

Materials

Stock basic fuchsin

MaterialAmount
Basic fuchsin
0.75g
Ethanol, 95%20mL
Distilled water80mL

Preparation

  1. Mix the ethanol and water. Add the basic fuchsin.
  2. Heat at 40°C for 48 hours. Cool and filter.

Solution A

MaterialAmount
Stock basic fuchsin5mL
Aniline water100mL
Acetic acid, glacial1.5mL
Distilled water100mL

Solution B

MaterialAmount
Formalin, conc. (40%)20mL
Acetic acid, glacial25mL
Distilled water50mL

Tissue Sample

5 µg 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 1-10 minute.
  3. Rinse with distilled water.
  4. Place into solution B for 5-10 minutes, until differentiated.
  5. Stain with a contrast stain.
  6. Dehydrate with ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  red
  • Cytoplasm  –  according to counterstain

Notes

  • Magenta is an older name for basic fuchsin. Since this is a mixture, it is likely that its component dyes could also 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.
    Citing:
    Duprès, (1935)
    Journal number 11425, v. 46, pp. 77

Note: Gray identifies journals by a number from the world list of scientific periodicals, ed. 2, 1934. Unfortunately, the journal listed as number 11425 is not identified by name in the list he provides. In a second reference to the differentiator used in this technique he gives the journal number as 14425. This may be the intended reference:
Duprès, (1935)
Monitore zoologico italiano, v. 46, pp. 77
Florence, Italy.

Proescher & Arkush Iron Alum-Celestine Blue for Nuclei

By Dye Type, Hematoxylin Alternatives, Protocols, Stain Type

Proescher & Arkush Iron Alum-Celestine Blue

for Nuclei

6
steps
4
materials

Materials

  • An eosin solution, or other counterstain
  • Iron alum-celestine blue
    MaterialAmount
    Ferric ammonium sulphate5g
    Celestine blue B0.5g
    Distilled water100mL

    Dissolve the ferric ammonium sulphate in the distilled water. Add the celestine blue B. Boil for 5 minutes. Cool and filter.

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 the iron alum-celestine blue solution for 3 minutes to 2 hours.
  3. Wash with tap water.
  4. Counterstain with eosin or other counterstain.
  5. Dehydrate with 95% and absolute ethanols.
  6. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Cytoplasm  –  pink

Notes

  • The iron alum-celestine blue solution is stable for a few months.
  • A staining time of 5-10 minutes is usually satisfactory for formalin fixed tissues.
  • This method has been recommended as a substitute for Hematoxylin and Eosin.
  • Proescher & Arkush also recommended the dyes gallamine blue and gallocyanin. Neither dye is as stable as celestine blue, although the color with gallocyanin most closely resembles alum hematoxylin of all three dyes.

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:
    Proescher and Arkush, (1928)
    Stain Technology, v. 3, pp. 36