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Methenamine Silver for Reducing Substances

By Intracytoplasmic Granules, Melanin & Enterochromaffin, Protocols, Stain Target
Protocol

Methenamine Silver

for Reducing Substances

13
steps
7
materials
print

Materials

  • Yellow gold chloride, 0.2% aqu.
  • Sodium thiosulfate, 3% aqu.
  • Stock Methenamine silver
    MaterialAmount
    Methenamine, 3% aqu.100mL
    Silver nitrate, 5% aqu.5mL

    Shake until the precipitate redissolves. Silvering of the container indicates deterioration.

  • Working Methenamine silver
    MaterialAmount
    Stock Methenamine silver50mL
    Borax, 5% aqu.5mL

    Make just before use and preheat to 56°C.

  • Mallory bleach
    MaterialAmount
    Potassium permanganate, 1% aqu.47.5mL
    Sulfuric acid, 3% aqu.2.5mL

Tissue Sample

5µ 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. Optionally, remove melanin with the Mallory bleach.
  3. Bleach in Oxalic acid for a few minutes.
  4. Rinse with distilled water.
  5. Treat with methenamine silver solution at 56&degC. until impregnated
  6. Wash with distilled water.
  7. Tone in gold chloride solution for 1 minute.
  8. Rinse with distilled water.
  9. Fix in sodium thiosulfate for 5 minutes.
  10. Wash well with running tap water.
  11. Counterstain with neutral red for 1 minute
  12. Rinse with tap water.
  13. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Melanin (unbleached) – Black
  • Melanin (bleached) – Unstained
  • Enterochromaffin – Black
  • Lipofuscin – Black
  • Nuclei – Red

Safety Note

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

References

  1. Histology Bench Manual.
    Prince George Regional Hospital
March 30, 2023
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Diazonium Reaction for Enterochromaffin

By Intracytoplasmic Granules, Melanin & Enterochromaffin, Protocols, Stain Target
Protocol

Diazonium Reaction

for Enterochromaffin

7
steps
3
materials
print

Materials

  • Mayer’s hemalum or similar
  • Solution A
    MaterialAmount
    Fast red B, 1% aqu.5mL
    Lithium carbonate, sat. aqu.2mL

    Refrigerate the stock solutions at 4°C. Use the working solution immediately it is prepared.

Tissue Sample

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


Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Rinse with distilled water.
  3. Treat with solution A for 1 minute at 4°C.
  4. Rinse well with distilled water.
  5. Stain with Mayer’s hemalum for 1 minute.
  6. Wash well with running tap water.
  7. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Enterochromaffin – Orange-red
  • Nuclei – Blue

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. (1977).
    Theory and practice of histological techniques.
    Churchill Livingstone, Edinburgh, UK.
March 30, 2023
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Langeron’s Stain for Cell Inclusions

By Intracytoplasmic Granules, Plasma Cells, Protocols, Stain Target
Protocol

Langeron's Stain

for Cell Inclusions

6
steps
7
materials
print

Materials

Stock solution A

MaterialAmount
Distilled water100mL
Methyl green4g
Phenol5g

Stock solution B

MaterialAmount
Distilled water100mL
Pyronin Y4g
Phenol5g

Working solution

MaterialAmount
Stock solution A25mL
Stock solution B25mL

Differentiator

MaterialAmount
Ethanol, absolute25mL
Acetone25mL

Tissue Sample

Most fixatives should be satisfactory.


Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into the staining solution for 15 minutes at 50°C.
  3. Rinse briefly with distilled water.
  4. Differentiate until staining is clear.
  5. Dehydrate with amyl alcohol.
  6. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei – violet
  • Plasma cell cytoplasm – red
  • Other cell cytoplasm – pink

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. p. 355
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
March 30, 2023
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Reddy’s Alum Hematoxylin

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

Reddy's Alum Hematoxylin

8
steps
5
materials
print

Materials

MaterialAmountFunction
Hematoxylin6.4 gDye
Ammonium alum60 gMordant
Ethanol, absolute200 mLSolvent
Glycerol160 mLStabiliser
Distilled water640 mLSolvent

Compounding Procedure

  1. Combine all the ingredients.
  2. Mix for a day.
  3. Leave in the dark for at least a month to ripen.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 3-5 minutes.
  3. Rinse with water.
  4. Differentiate lightly with 1% hydrochloric acid in 70% ethanol.
  5. Rinse with water.
  6. Blue with 0.1% sodium bicarbonate.
  7. Wash with water.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei – blue

Safety Note

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

References

  1. Reddy, P. H., (2001)
    Neurological Sciences Institute
    Oregon Health and Science University
March 30, 2023
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Crétin’s Iron Hematoxylin

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

Crétin's Iron Hematoxylin

8
steps
6
materials
print

Materials

Solution A

MaterialAmountFunction
Ferrous sulfate4 gMordant
Distilled water100 mLSolvent

Solution B

MaterialAmountFunction
Potassium ferrocyanide2 gMordant
Potassium ferricyanide1 gMordant
Distilled water100 mLSolvent

Solution C

MaterialAmountFunction
Hematoxylin0.5 gDye
Distilled water100 mLSolvent

Solution D

MaterialAmountFunction
Ferric ammonium sulfate5 gMordant
Distilled water100 mLSolvent

Compounding procedure

Make each solution seperately.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into solution A for 24 hours.
  3. Wash with running tap water overnight.
  4. Place into solution B for 3-6 hours.
  5. Rinse with distilled water.
  6. Place into solution C overnight.
  7. Place into solution D until differentiated.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei – dense black

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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Celestine Blue Hemalum An Acid Resistant Nuclear Stain

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

Celestine Blue Hemalum

An Acid Resistant Nuclear Stain

6
steps
2
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 the iron alum-celestine blue solution for 10 minutes.
  3. Wash with tap water.
  4. Place into the alum hematoxylin for 10 minutes.
  5. Wash with water to blue.
  6. Continue with the counterstain

Expected Results

  • Nuclei – blue-black
  • Other tissue – according to the counterstain

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. Bancroft, J.D. and Stevens A. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
  3. Humason, G. L., (1967)
    Animal tissue techniques, Ed. 2
    W. H. Freeman and Company, San Francisco, Ca, USA
  4. Drury, R.A.B. and Wallington, E.A., (1980)
    Carleton’s histological technique Ed. 5
    Oxford University Press, Oxford, UK.
  5. Putt, F. A.
    Manual of Histopathological Staining Methods
    John Wiley & Sons, New York, NY., USA
March 30, 2023
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Hotchkiss’ Alcoholic Periodic Acid Schiff

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

Hotchkiss' Alcoholic Periodic Acid Schiff

13
steps
10
materials
print

It was initially thought that the periodic acid Schiff reaction could result in less glycogen being demonstrated than was actually present because it might dissolve in aqueous reagents. It is now known this is not a concern. Hotchkiss recommended an alcoholic method to ensure it did not take place. This method is now redundant.

Materials

  • Schiff’s reagent
  • Mayer’s hemalum
  • Alcoholic periodic acid
    MaterialAmount
    Periodic acid0.8g
    Sodium acetate buffer 0.2M10mL
    Ethanol, absolute70mL
    Distilled water20mL
  • Acid reducing rinse
    MaterialAmount
    Potassium iodide2g
    Sodium thiosulphate2g
    Ethanol absolute60mL
    Hydrochloric acid N/12mL
    Distilled water40mL

Tissue Sample

Presumably an alcoholic fixative should be required if glycogen were to dissolve in aqueous solutions. However, 5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are usually satisfactory, although glutaraldehyde should be avoided.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into periodic acid for 10 minutes.
  3. Rinse with 70% ethanol.
  4. Place in acid reducing rinse for 1 minute.
  5. Rinse with 70% ethanol.
  6. Wash with running water to remove ethanol.
  7. Rinse with distilled water.
  8. Place in Schiff’s reagent for 10-30 minutes.
  9. Wash off with distilled water.
  10. Wash well with tap water for about 10 minutes.
  11. Counterstain with Mayer’s hemalum for 1 minutes.
  12. Wash well with tap water until hemalum is blued.
  13. Dehydrate with ethanol, clear with xylene, and coverslip using a resinous medium.

Expected Results

  • Oxidisable carbohydrates – red
  • Nuclei – blue

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., (1963)
    Handbook of histopathological and histochemical techniques Ed. 2
    Butterworth, London, UK.
March 30, 2023
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Fluorescent Nucleal Reaction for DNA

By Aldehydes, Nucleal Reaction, Protocols, Schiff's Reagent Reactions, Stain Target, Stain Type
Protocol

Fluorescent Nucleal Reaction

for DNA

9
steps
3
materials
print

Materials

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Many other fixatives are satisfactory. Fixatives containing strong acids should be avoided as this method depends on the acid hydrolysis of DNA, and acids in some fixatives may pre-hydrolyse the tissue (picric acid in Bouin’s aqueous formal-picric-acetic mixture for example).

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Rinse briefly with cold 1 N hydrochloric acid.
  3. Place into prewarmed hydrochloric acid for the appropriate time at 60°C.
  4. Rinse briefly with cold 1 N hydrochloric acid.
  5. Rinse briefly with distilled water.
  6. Place into Acriflavine Schiff’s reagent for 30-60 minutes at room temperature.
  7. Place into 1% acid alcohol, 2 changes for about 5 minutes each.
  8. Wash well with water.
  9. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Using a BG 12 exciter filter, and OG 4 (yellow) and/or OG5 (orange) barrier filter, DNA fluoresces yellow.

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, Barr, W T, (1985).
    Cellular pathology technique., Ed. 4, p. 189.
    Butterworths, London, England.
March 30, 2023
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Pseudo-Periodic Acid Schiff

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

Pseudo-Periodic Acid Schiff

10
steps
2
materials
print

The terms Pseudo-Schiff and Pseudo-PAS refer to the use of dyes other than pararosanilin or basic fuchsin to make a Schiff type solution and use it for demonstrating carbohydrates. While an interesting exercise, it has little practical use except for the occasional demonstration of fungi with a fluorescent pseudo-Schiff solution made with acriflavine. The following dyes, among others, have been suggested.

DyeCI NumberColor
Acid fuchsin42685violet
Acriflavine46000yellow
Azure A52005blue
Azure C52005blue
Crystal violet42555blue-violet
Methyl violet42535violet
Methylene blue52015blue
Safranin O50240red
Thionin52005blue
Toluidine blue52040blue

Materials

  • 1% hydrochloric acid in 70% ethanol
  • A suitable counterstain

Preparation of Pseudo-Schiff Solution

Prepare a solution according to the instructions for acriflavine Schiff reagent, or another formula, if preferred. Note that they will likely not become colourless, so leave for 48 hours, then filter the solution and store refrigerated.

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are usually satisfactory, although glutaraldehyde should be avoided.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into periodic acid for 10 minutes.
  3. Wash with running water.
  4. Rinse with distilled water.
  5. Place in pseudo-Schiff’s reagent for 10-30 minutes.
  6. Rinse with distilled water.
  7. Place in acid ethanol for 5 minutes.
  8. Wash well with tap water for about 10 minutes.
  9. Counterstain with a suitable contrasting nuclear stain.
  10. Dehydrate with ethanol, clear with xylene and coverslip using a resinous medium.

Expected Results

  • Oxidisable carbohydrates – coloured according to the dye used
  • Nuclei – as stained

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., (1963)
    Handbook of histopathological and histochemical techniques Ed. 2
    Butterworth, London, UK.
March 30, 2023
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Slidders’ Orange-Fuchsin-Green for Pituitary Cells

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

Slidders' Orange-Fuchsin-Green (OFG)

For Pituitary Cells

14
steps
10
materials
print

Materials

  • Lendrum’s celestine blue
  • A progressive hemalum, such as Mayer’s
  • Solution A
    MaterialAmount
    Orange G0.05g
    Ethanol, 95%100mL
    Phosphotungstic acid0.5g
  • Solution B
    MaterialAmount
    Acid fuchsin0.5g
    Acetic acid, glacial0.5mL
    Distilled water100mL
  • Solution C
    MaterialAmount
    Phosphotungstic acid1g
    Distilled water100mL
  • Solution D
    MaterialAmount
    Light green SF yellowish1.5g
    Acetic acid, glacial1.5mL
    Distilled water100mL

Tissue Sample

Although 5µ paraffin sections of neutral buffered formalin fixed tissue are likely adequate, superior results are obtained with fixation in formal sublimate, preferably about 48 hours. Other fixatives may be satisfactory. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Remove mercury pigment.
  3. Stain nuclei with Lendrum’s celestine blue-hemalum.
  4. Rinse with 95% ethanol.
  5. Place into solution A for 2 minutes.
  6. Rinse well with distilled water
  7. Place into solution B for 2-5 minutes, until basophiles are red.
  8. Rinse with distilled water.
  9. Differentiate with solution C for 5 minutes.
  10. Rinse with distilled water.
  11. Place into solution D for 2 minutes
  12. Rinse well with distilled water.
  13. Dehydrate with ethanols.
  14. Clear with xylene and mount with a resinous medium.

Expected Results

  • Best results are obtained if staining times are controlled visually.

Notes

  • Nuclei – black
  • Erythrocytes – orange
  • Collagen – green
  • Acidophils – orange
  • Basophils – red
  • Chromophobes – pale gray-green

Safety Note

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

References

  1. Putt, F. A.
    Manual of Histopathological Staining Methods
    John Wiley & Sons, New York, NY., USA
    Citing:
    Slidders, W. J., (1961)
    Journal of Pathology and Bacteriology, v. 82, p. 532
    London, England.
March 30, 2023
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