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Stain Target

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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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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Walter’s Trichrome for Elastic and Collagen

By Dye Type, Elastic Fibers, Orcein, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, One-Step
Protocol

Walter's Trichrome

for Elastic and Collagen

9
steps
8
materials
print

Materials

Solution A

MaterialAmount
Ferric ammonium sulphate2.5g
Distilled water100mL

Solution B

MaterialAmount
Phosphotungstic acid2g
Distilled water100mL

Solution C

MaterialAmount
Eosin B0.7g
Acid fuchsin, sat. aqu4mL
Unna’s elastic stain35mL
Ethanol, 50%35mL
Glycerol40mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. 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. Place into solution A for 24 hours.
  3. Rinse quickly with distilled water.
  4. Place into solution B for 10 minutes.
  5. Rinse quickly with distilled water.
  6. Place into solution C for 15-20 minutes.
  7. Rinse quickly with 95% ethanol until clouds of dye stop being extracted.
  8. Dehydrate with 100% ethanol for 1 minute.
  9. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  purple
  • Elastic fibres  –  purple
  • Erythrocytes  –  orange
  • Collagen  –  blue

Notes

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:
    Walter, (1930)
    Zeitschrift für wissenschaftliche Mikroskopie und für mikroskipische Technik,
    v. 46, pp. 458
March 30, 2023
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Gomori’s Impregnation for Reticulin

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

Gomori's Impregnation

for Reticulin

19
steps
9
materials
print

Materials

  • Silver nitrate, 2% aqu.
  • Silver nitrate, 20% aqu.
  • Strong ammonium hydroxide (s.g. 0.88).
  • Sodium hydroxide, 40% aqu.
  • Periodic acid, 0.5% aqu.
  • Formalin, 3% aqu.
  • Yellow gold chloride, 0.5% aqu.
  • Sodium thiosulphate, 5% aqu.
  • Neutral red, 1% aqu.

Preparation of Ammoniacal Silver

  1. Place 10 mL of 10% silver nitrate in a flask.
  2. Add 2.5 mL of 10% potassium hydroxide.
  3. Allow the precipitate to settle then remove the supernatent with a Pasteur pipette.
  4. Wash the precipitate twice with distilled water, allowing the precipitate to settle and draining after each.
  5. Add 10 mL distilled water.
  6. While swirling, slowly add drops of strong ammonium hydroxide until the precipitate just redissolves.
  7. Slowly add a drop or more of 10% silver nitrate until the solution becomes very faintly opalescent.
  8. Make up to 20 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. Oxidise with 1% potassium permanganate for 2 minutes.
  3. Bleach in Oxalic acid for a 2 minutes.
  4. Rinse well with tap water.
  5. Sensitise with 2.5% iron alum solution for 1 minute.
  6. Rinse with tap water.
  7. Rinse well with distilled water.
  8. Treat with ammoniacal silver for 1 to 3 minutes.
  9. Rinse briefly with distilled water.
  10. Reduce in 10% formalin for 3 minutes.
  11. Rinse well with tap water.
  12. Rinse with distilled water.
  13. Tone with 0.2% gold chloride solution.
  14. Rinse with distilled water.
  15. Fix in 5% sodium thiosulphate for 5 minutes.
  16. Wash well with running tap water.
  17. Counterstain with neutral red for 1 minute.
  18. Rinse with tap water.
  19. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Nuclei  –  red
  • Background  –  grey

Notes

  • Ensure that both the ammonium hydroxide and potassium hydroxide are fresh and full strength. Keep both well stoppered when not in use. For the ammonium hydroxide, pour sufficient for use from the stock bottle into a beaker, then immediately restopper the stock bottle. Do not return excess ammonium hydroxide to the stock bottle.
  • After making the ammoniacal silver solution, smell the solution to ensure it has only a faint smell of ammonia. If the smell of ammonia is strong it indicates that too much ammonium hydroxide has been added. If so, it is preferable to make the solution again. Improperly made ammoniacal silver solutions can affect the quality of the impregnation.
  • Drury & Wallington say to bleach with 3% aqueous potassium metabisulphite instead of oxalic acid at step 3.
  • Drury & Wallington also specify treatment with 3% aqueous potassium bisulphite for one minute followed by a distilled water rinse, immediately after toning (step 13). They do not say if this is part of the toning procedure or is an independent step. Culling et. al. omit it.
  • Iron alum is ferric ammonium sulphate.
  • 10% formalin is made by diluting strong formalin 1:10 with tap water (10 mL strong formalin, 90 mL tap water).
  • Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black reticulin fibres on a pale grey-brown background. Others tone longer (a few minutes) to produce black reticulin fibres on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.

Safety Note

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

References

  1. Drury, R A, and Wallington, E A, (1967).
    Carleton’s histological technique., Ed. 5.
    Oxford University Press, London, England.
  2. Culling, C F A, Allison, R T, Barr, W T, (1985).
    Cellular pathology technique., Ed. 4.
    Butterworths, London, England.
March 30, 2023
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Bancroft’s Crystal Violet for Amyloid

By Amyloid, Metachromasia, Protocols, Stain Target, Stain Type
Protocol

Bancroft's Crystal Violet

for Amyloid

7
steps
3
materials
print

Materials

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Cryostat sections usually show brighter metachromasia. Unmounted frozen sections may also be floated in each solution and mounted on a slide just before coverslipping.

Protocol

  1. Bring sections to water via xylene and ethanol, except for cryostat and frozen sections.
  2. Place into crystal violet solution for 5 minutes.
  3. Rinse with distilled water.
  4. Differentiate briefly with dilute acetic acid for 15 – 20 seconds.
  5. Counterstain with methyl green for 5 – 15 minutes.
  6. Wash with distilled water.
  7. Either drain all water from the slide until just damp then blot and mount with Apathy’s or Highman’s medium, or drain all water from the slide until just damp then blot and flood with xylene. Repeat until the section is cleared, then mount with a resinous medium.

Expected Results

  • Amyloid – purple-red
  • Background – green
  • Nuclei – green

Safety Note

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

References

  1. Bancroft, J. D., (1963).
    Stain technology, v. 38, p. 336.London, England.
March 30, 2023
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Bancroft’s Methyl Green for Amyloid

By Amyloid, Metachromasia, Protocols, Stain Target, Stain Type
Protocol

Bancroft's Methyl Green

for Amyloid

6
steps
2
materials
print

Materials

  • Methyl green, 2% aqueous, washed with chloroform to remove crystal violet
  • Acetic acid, 1% aqueous

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Cryostat sections usually show brighter metachromasia. Unmounted frozen sections may also be floated in each solution and mounted on a slide just before coverslipping.

Protocol

  1. Bring sections to water via xylene and ethanol, except for cryostat and frozen sections.
  2. Place into methyl green solution for 1-5 minutes.
  3. If necessary, differentiate in dilute acetic acid until amyloid is red and contrasts well.
  4. Rinse well with tap water.
  5. Drain all water from the slide until just damp and blot dry.
  6. Flood with triethylphosphate, then with xylene, and coverslip using a resinous medium.

Expected Results

  • Amyloid – purple-red
  • Background – green
  • Nuclei – green

Safety Note

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

References

  1. Drury, R A, and Wallington, E A, (1967).
    Carleton’s histological technique., Ed. 4, p. 222.
    Oxford University Press, London, England.
March 30, 2023
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