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Vassar & Culling’s Thioflavine T for Amyloid Fluorescence

By Amyloid, Fluorescent Staining, Protocols, Stain Target, Stain Type
Protocol

Vassar & Culling's Thioflavine T

for Amyloid Fluorescence

8
steps
3
materials
print

Materials

Tissue Sample

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

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into alum hematoxylin for 2 minutes.
  3. Rinse well with water.
  4. Place into thioflavine T solution for 3 minutes.
  5. Rinse with water.
  6. Place into acetic acid solution for 20 minutes.
  7. Wash with water.
  8. Mount in a fluorescence free aqueous mounting medium.

Expected Results

Using a UG1 or UG2 exciter filter and a UV barrier filter, or a BG12 exciter and an OG4 or OG5 barrier filter, amyloid fluoresces bright yellow.

Notes

  • The pretreatment with alum hematoxylin suppresses nuclear fluorescence.
  • Some workers have reported that materials other than amyloid may fluoresce yellow. The authors say this is caused by using a yellow barrier filter and strongly recommended the first filter combination. With this, these materials fluoresce white or pale blue.

Safety Note

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

References

  1. Vassar, P.S. and Culling, C.F.A., (1959),
    Fluorescent stains with special reference to amyloid and connective tissue,
    Archives of pathology, v 68, page 487
  2. 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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Wolman’s Standard Toluidine Blue (STB) for Amyloid

By Amyloid, Protocols, Stain Target
Protocol

Wolman's Standard Toluidine Blue(STB)

for Amyloid

7
steps
3
materials
print

Materials

MaterialAmount
Toluidine blue1g
Distilled water50mL
Iso-propanol, absolute50mL

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 in the toluidine blue solution at 37°C for 30 minutes.
  3. Blot carefully.
  4. Place into absolute iso-propanol for one minute.
  5. Blot carefully.
  6. Clear with xylene and coverslip using Canada balsam.
  7. Examine microscopically using crossed polarizing filters.

Expected Results

  • Amyloid – orange-red to red birefringence.
  • Orthochromatic tissue – blue-white birefringence
  • metachromatic tissue – yellow-green birefringence

Notes

  • Wolman strongly recommended this procedure, considering it to be highly selective for amyloid.
  • The birefringence is independent of section thickness and the quality of the microscope optics.

Safety Note

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

References

  1. Wolman, M. (1971).
    Amyloid, its nature and molecular structure: comparison of a new toluidine blue polarized light method with traditional procedures.
    Laboratory Investigation, v. 25: p. 104-110.
March 30, 2023
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Birch-Hirschfeld’s Crystal Violet for Amyloid

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

Birch-Hirschfeld's Crystal Violet

for Amyloid

9
steps
4
materials
print

Materials

Tissue Sample

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

Protocol

  1. Bring sections to water via xylene and ethanol, except for cryostat and frozen sections.
  2. Place into bismarck brown solution for 5 minutes.
  3. Rinse well with 95% ethanol, then rinse with distilled water.
  4. Place into crystal violet solution for 5 minutes.
  5. Rinse with water.
  6. If necessary, differentiate in 1% acetic acid until amyloid is red and contrasts well with the tissue.
  7. Wash well in tap water.
  8. Drain all water from the slide until just damp and mount with levulose syrup.
  9. Ring the coverslip to inhibit evaporation of the mounting medium.

Expected Results

  • Amyloid  –  purple-red
  • Background  –  blue-violet
  • Nuclei  –  brown

Notes

  • Methyl violet may be used instead of crystal violet if preferred.
  • Although levulose syrup (fructose syrup or high fructose corn syrup) is specified it is likely that Highman’s gum syrup would be preferable as it inhibits leaching of the dye.
  • Although bismarck brown is metachromatic (yellow metachromasia), it is used here as a basic dye for staining nuclei.

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.451.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
March 30, 2023
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Jurgens’ Crystal Violet for Amyloid

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

Jurgens' 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 2-5 minutes.
  3. Rinse well with water and examine microscopically.
  4. If necessary, differentiate in dilute acetic acid until amyloid is red and contrasts well with the tissue.
  5. Wash very well in tap water, about 5 minutes.
  6. Drain all water from the slide until just damp and mount with Highman’s medium.
  7. Ring the coverslip to inhibit evaporation of the mounting medium and precipitation of the ingredients.

Expected Results

  • Amyloid  –  purple-red
  • Background  –  blue-violet
  • Nuclei  –  blue-violet

Notes

  • Methyl violet may be used instead of crystal violet if preferred.
  • Highman’s gum syrup is a modification of Apathy’s gum syrup and contains potassium acetate or sodium chloride to stop bleeding of the dye into the mounting medium.

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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Anderson’s Alum Hematoxylin

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

Anderson's Alum Hematoxylin

8
steps
7
materials
print

Although there are three variations listed for Anderson’s formulas, those marked as 1923a and 1923b are variations of the same solution, the former being that given in the Microtomist’s Formulary and Guide and the latter in the Microtomist’s Vade Mecum.

Materials

MaterialVariantFunction
1923a1923b1929
Hematoxylin2.5 g2.5 g5 gDye
Ammonium alum20 gSat.30 gMordant
Distilled water900 mL900 mL700 mLSolvent
95% ethanol50 mL50 mL50 mLSolvent
Calcium hypochlorite4 g–40 gOxidant
Chloramine T or lime chloride4 gOxidant
Glacial acetic acid50 mL50 mL50 mLAcidifier

Compounding procedures

1923a & 1929

  1. Dissolve the calcium hypochlorite in 200 mL water.
  2. Dissolve the hematoxylin in some of the water.
  3. After 4 hours, combine the solutions.
  4. Dissolve the other ingredients in the rest of the water.
  5. Combine with the hematoxylin-hypochlorite solution.

1923b

  1. Bring 700 mL water to a boil, then saturate it with alum (see notes).
  2. Allow to cool for one day, then filter.
  3. Dissolve the chloramine T or lime chloride into 200 mL of water.
  4. Leave four hours. Shake occasionally.
  5. Dissolve the hematoxylin in the ethanol
  6. Add the oxidant solution to the hematoxylin.
  7. Mix for a few seconds. It should be dark brown.
  8. Slowly add to the 700 mL alum solution, while shaking.
  9. Add the acetic acid.
  10. It is ready immediately.

Protocol

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

  • The 1923a formula oxidizes 2.5 grams hematoxylin with 4 grams calcium hypochlorite. This is a ratio of 1.6:1.
  • The 1929 formula oxidizes 5 grams hematoxylin with 40 grams calcium hypochlorite. This is a ratio of 8:1.
  • The 1923b formula is an alternate formula given by Bolles-Lee. It differs from the 1923a formula by using a saturated alum solution, and chloramine T or “commercial chloride of lime” (a crude preparation of calcium chloride with other substances present) as oxidant.
  • The 1923b formula calls for 700 mL saturated aqueous alum. The instructions specify that the water should be saturated at boiling, then cooled to room temperature. This would require about 350 grams alum (at 500 mg/mL), but at room temperature, the solution would contain only about 100 grams (at 150 mg/mL). Dissolving 110 grams alum in 700 mL hot water, cooling and filtering would give the same solution.
  • 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. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
  2. Bolles Lee, A.. Edited by Gatenby, J.B. and Beams, H.W., (1950)
    The Microtomist’s Vade-Mecum. 11 ed.,
    Churchill, London, UK.
    Citing:
    Anderson, J., (1923)
    Journal of Pathology and Bacteriology.
  3. Susan Budavari, Editor, (1996)
    The Merck Index, Ed. 12
    Merck & Co., Inc., Whitehouse Station, NJ, USA
March 30, 2023
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Anderson’s Iron Hematoxylin

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

Anderson's Iron Hematoxylin

6
steps
7
materials
print

Materials

Solution A

MaterialAmountFunction
Hematoxylin0.5 gDye
100% ethanol50 mLSolvent
Distilled water50 mLSolvent
Calcium hypochlorite, 2% aqueous5 mLOxidant

Solution B

MaterialAmountFunction
Ferric ammonium sulphate3 gMordant
Distilled water100 mLSolvent
Sulfuric acid0.5 mLAcidifier

Compounding procedures

  1. Make each solution separately.
  2. For use, add 2 volumes of solution A to 1 volume of solution B.
  3. The working solution may be used immediately, but is not stable for long.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  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.

Expected Results

  • Nuclei  –  black
  • Background  –  as counterstain or unstained

Notes

  • The stock solutions are stable for some time.
  • The working solution should be made fresh.
  • It is claimed that the working solution rarely overstains, i.e. it is progressive.
  • Solution A incorporates calcium hypochlorite as an oxidizing agent for hematoxylin. Presumably other oxidizing agents would suffice equally as well. Sodium iodate (0.1 g or less) is the most common.
  • The staining time should be determined by trial.

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:
    Anderson, J., (1929)
    How to stain the nervous system.
    Livingstone. Edinburgh, Scotland.
March 30, 2023
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Apathy’s Alum Hematoxylin

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

Apathy's Alum Hematoxylin

8
steps
7
materials
print

Materials

MaterialAmountFunction
Hematoxylin3 gDye
Ammonium alum30 gMordant
Distilled water450 mLSolvent
95% ethanol250 mLDye solvent
Glycerol350 mLStabiliser
Glacial acetic acid10 mLAcidifier
Salicylic acid0.3 gAcidifier

Compounding procedure

  1. Dissolve the dye in 100 mL water and 250 mL ethanol.
  2. Leave at room temperature to ripen (months).
  3. Dissolve the alum and acids in 350 mL water.
  4. Combine both solutions.
  5. Add glycerol.

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

  • Lillie excludes the salicylic acid.
  • The appropriate staining time should be determined by trial.
  • 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. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
  2. Lillie, R.D., (1954)
    Histopathologic technique and practical histochemistry Ed.2
    Blakiston, New York, USA.
March 30, 2023
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Roach & Smith’s Bismuth Hematoxylin

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

Roach & Smith's Bismuth Hematoxylin

4
steps
7
materials
print

This method demonstrates arginine.

Materials

MaterialAmountFunction
Hematoxylin50 mgDye
Ethanol, abs.5 mLSolvent
Sodium iodate10 mgOxidant
Bismuth nitrate50 mgMordant
Citric acid, 0.5M aqu.85 mLAcidifier
Glycerol20 mLSolvent
Sodium hydroxide, 1 M aqu.19 mLAlkaliser

Compounding procedure

  1. Dissolve the bismuth nitrate into the citric acid solution.
  2. Add 10 mL of the glycerol, then the sodium hydroxide solution.
  3. Adjust the pH to 5.2.
  4. Dissolve the hematoxylin into the ethanol.
  5. Combine the two solutions.
  6. Add the sodium iodate dissolved in 1 mL distilled water.
  7. Stir for 20 minutes.
  8. Add the remaining 10 mL of glycerol.

This solution has a usable life of about 3 hours.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into bismuth hematoxylin for 10 minutes.
  3. Rinse with water.
  4. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Arginine residues  –  black

Notes

  • The formula calls for bismuth nitrate pentahydrate, Bi(NO3)3.5H20.
  • This method demonstrates proteins with an arginine content of about 12% or more.
  • Nuclear histones and myelin basic protein stain strongly.
  • The authors suggest that the bismuth binds to the guanidine groups of arginine.

Safety Note

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

References

  1. Roach, J. B., and Allen, A. A., (1997)
    Bismuth hematoxylin stain for arginine residues.
    Biotechnic & Histochemistry, v.72, Nº 1, p. 49.
March 30, 2023
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Proescher & Arkush Iron Alum-Celestine Blue for Nuclei

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

Proescher & Arkush Iron Alum-Celestine Blue

for Nuclei

6
steps
4
materials
print

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
March 30, 2023
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Gram Churukian–Schenk for Gram Positive & Negative Bacteria

By Bacteria, Gram Staining, Protocols, Stain Target, Stain Type
Protocol

Gram Churukian–Schenk

for Gram Positive & Negative Bacteria

14
steps
15
materials
print

Materials

  • Stock basic fuchsin
    MaterialAmount
    Basic fuchsin0.5g
    Distilled water100mL
  • Solution A
    Crystal violet 10% in 2mL ethanol

    MaterialAmount
    Ammonium oxalate 1% aqueous98mL
  • Solution B
    MaterialAmount
    Iodine2g
    Potassium iodide4g
    Distilled water400mL
  • Solution C
    MaterialAmount
    Absolute ethanol1volume
    Acetone1volume
  • Solution D
    MaterialAmount
    Stock basic fuchsin5mL
    Distilled water45mL
  • Solution E
    MaterialAmount
    Picric acid0.1g
    Acetone100mL
  • Solution F
    MaterialAmount
    Acetone1Volume
    Xylene1volume

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 in solution A for 2 minutes.
  3. Rinse with tap water.
  4. Place in solution B for 1 minute.
  5. Rinse well with tap water.
  6. Blot the slide, but not the tissue.
  7. Decolorise with solution C until no more blue floods off.
  8. Wet section with solution D then apply for 1 minute.
  9. Rinse with distilled water.
  10. Blot the slide, but not the tissue.
  11. Place in acetone for 3 seconds.
  12. Differentiate in solution E for 10 seconds.
  13. Quickly dip a few times in solution F.
  14. Clear with xylene and mount with a resinous medium.

Expected Results

  • Gram positive bacteria  –  blue
  • Nocardia and actinomyces  –  blue, or blue and red
  • Gram negative bacteria  –  red
  • Nuclei, Elastic, Paneth cells  –  red
  • Background  –  yellow

Notes

  • Picric acid should be handled with care. Solution E may be made by taking 12 mL of a saturated solution of picric acid in ethanol and diluting to 1 liter with acetone.
  • Basic fuchsin homologues with CI numbers of 42500 (pararosanilin) or 42510 (rosanilin) were specified. It was also noted that new fuchsin (CI 42520) was satisfactory, but not recommended because it was not certified by the Biological Stain Commission.
  • The authors note that sections must not be allowed to dry out after being stained with basic fuchsin. Doing so makes it difficult or impossible to properly differentiate the red counterstain.

Safety Note

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

References

  1. Churukian, C. J. & Schenk, E. A. (1982)
    A method for demonstrating Gram-positive and Gram-negative bacteria.
    Journal of Histotechnology, v.5, No.3, p.127
March 30, 2023
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