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Weigert’s Iron Hematoxylin

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

Weigert's Iron Hematoxylin

8
steps
8
materials
print

Materials

Solution A

MaterialVariantFunction
19031904
Ferric chloride0.4 g0.6 gMordant
Distilled water100 mL100 mLSolvent
Hydrochloric acid–0.75 mLSolvent

Solution B

MaterialVariantFunction
19031904
Hematoxylin1 g1 gDye
95% ethanol100 mL100 mLSolvent

Solution C

MaterialVariantFunction
19031904
Potassium ferricyanide2.5 g–Bleach
Sodium borate2 g–Alkaliniser
Distilled water100 mL–Solvent

Note: The 1904 formula is the solution usually meant when Weigert’s iron hematoxylin is specified as an acid resistant nuclear stain.

Compounding Procedure

For both variants:

  1. Make each solution separately.
  2. Filter.
  3. Immediately before use, combine equal parts of solutions A and B.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 1-2 hours.
  3. Rinse with tap water.
  4. 1903 – Place in solution C until differentiated.
    1904 – 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

  • The stock solutions are stable for some time.
  • The working solution should be made fresh.
  • The alcoholic hematoxylin solution was originally made by diluting a stock solution of 10% hematoxylin in 95% ethanol.
  • Solution A of the 1904 variant was originally made from a commercial ferric chloride solution, the amounts above being given by Gray based on its formula. The following is now usually specified:30% aqueous ferric chloride – 4 mL

    Distilled water – 100 mL

    Hydrochloric acid – 1 mL

  • Both methods were originally intended to be used without a counterstain for demonstrating chromatin and other structures usually stained by iron hematoxylin. If the 1904 formula is used for this purpose, the staining time should be increased and the excess stain removed with acid ethanol.

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:–
    Ehrlich, P., Krause, R. et. al., (1910)
    Enzyklopädie der mikroskopischen technik, ed. 2, v. 1, p. 231.
    Weigert, K., (1904)
    Zeitschrift für wissenschaftliche mikroskopie und für mikroskopische technik,
    v. 21, p. 1.
  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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Weiss’ Trichrome for Muscle and Collagen

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

Weiss' Trichrome

for Muscle and Collagen

8
steps
7
materials
print

Materials

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. Stain with an acid resistant nuclear stain.
  3. Place into solution A for 1 minute.
  4. Drain.
  5. Place into solution B for 4 minutes.
  6. Wash briefly with distilled water.
  7. Dehydrate with ethanol.
  8. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Erythrocytes  –  yellow
  • Cytoplasm  – red
  • Collagen  –  Blue

Notes

  • The original recommended nuclear staining in Delafield’s alum hematoxylin, with blueing.
  • This differs from Brillmeyer’s trichrome by using a weaker acid fuchsin solution and staining in aniline blue for a much shorter time.

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:
    Weiss, (1932)
    Stain Technology, v. 7, pp. 131
March 30, 2023
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Yasvoyn’s Iron Hematoxylin

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

Yasvoyn's Iron Hematoxylin

5
steps
3
materials
print

Materials

Solution A

MaterialAmountFunction
Hematoxylin0.1 gDye
Distilled watermLSolvent

Solution B

MaterialAmountFunction
Ferric ammonium sulfate2.5 gMordant
Distilled water100 mLSolvent

Compounding Procedure

  1. Make each solution separately.
  2. For use, add solution B drop by drop to 20 drops of solution A until it just remains blue .
  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 2-5 minutes.
  3. Rinse with water.
  4. Remove excess stain with 70% ethanol if necessary.
  5. 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.
  • Although not specified, a tap water wash to blue the hematoxylin may be useful following step 4.
  • Following a wash to blue the nuclei, a counterstain could probably be applied, if wished, before dehydration.

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:
    Roskin, G.E, (1946).
    Mikroskopecheskaya technica, p. 150
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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Highman’s Crystal Violet for Amyloid

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

Highman's Crystal Violet

for Amyloid

7
steps
5
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. Stain nuclei with Weigert’s iron hematoxylin for 5 minutes.
  3. Wash with water.
  4. Place into crystal violet solution for 1-30 minutes until amyloid is stained.
  5. Rinse well with water.
  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 – black

Notes

  • Methyl violet may be used instead of crystal violet if preferred.
  • Gray notes that Lieb substituted a solution of 0.3% crystal violet in 0.3% hydrochloric acid for Highman’s crystal violet solution.
  • 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. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide, p. 452.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
March 30, 2023
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Lendrum’s’ Methyl Violet for Amyloid

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

Lendrum's' Methyl 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 methyl violet solution for 3 minutes.
  3. Differentiate in formalin until amyloid is red and contrasts well with the tissue.
  4. Place into sodium chloride solution for 5 minutes.
  5. Rinse well with tap water.
  6. Drain all water from the slide until just damp and mount with corn syrup.
  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

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), p. 466.
    Cellular pathology technique. Ed. 4.,
    Butterworths, London, England.
March 30, 2023
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King’s Silver Impregnation for Amyloid

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

King's Silver Impregnation

for Amyloid

7
steps
10
materials
print

Materials

  • Pyridine
  • Ethanol 50%, 75%, 95%
  • Sodium carbonate
    MaterialAmount
    Sodium carbonate, anhydrous3.5g
    Distilled water100mL
  • Silver nitrate, 10%
    MaterialAmount
    Silver nitrate10g
    Distilled water100mL
  • Carbol xylene
    MaterialAmount
    Xylene3volumes
    Phenol1volume
  • Ammoniacal Silver
    MaterialAmount
    Silver nitrate, 10% aqueous5mL
    Strong ammonia (S.G. 0.880)asrequired
    Sodium carbonate, aqueous6.8mL
    Distilled waterasrequired

    Add ammonium hydroxide drop by drop to 5 mL of the silver solution until the precipitate which forms just redissolves. Add 6.8 mL sodium carbonate solution and sufficient water to make up to 75 mL. For use, add a few drops of pyridine to each 10 mL of the solution.

Tissue Sample

10-15µ free floating frozen sections of formalin fixed tissues. Collect into water and wash in a few changes of distilled water to remove residual formalin.


Protocol

  1. Add pyridine to 10 mL ammoniacal silver solution and warm to 40°C.
  2. Float sections in the warm ammoniacal silver until they turn brown.
  3. Rinse with water.
  4. Place in 50% ethanol for 5-10 minutes.
  5. Quickly treat with 75% and 95% ethanols.
  6. Clear with carbol xylene.
  7. Mount sections on to slides and coverslip, using a resinous medium.

Expected Results

  • Amyloid – brown to black.

Safety Note

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


References

  1. King, L.S., (1948),
    Atypical Amyloid Disease: With Observation on a New Silver Stain for Amyloid, American Journal of Pathology, v 24, page 1095-1115
March 30, 2023
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Lynch & Inwood’s Gold for Amyloid

By Amyloid, Metal Impregnation, Metal Impregnation, Non-Silver, Protocols, Stain Target, Stain Type
Protocol

Lynch & Inwood's Gold

for Amyloid

7
steps
5
materials
print

Materials

  • Chloro-auric acid, 1% aqueous
  • Hydrogen peroxide, 3% aqueous
  • Iodine solution
    MaterialAmount
    Iodine1g
    Potassium iodide2g
    Distilled water100mL

    Mix the potassium iodide and iodine crystals together. Add 5 mL of the water and mix until both have dissolved. Add the rest of the water.

Tissue Sample

5-10µ sections from formalin fixed, paraffin embedded tissues, mounted on slides are suitable.


Protocol

  1. Bring sections to water through xylene and ethanols.
  2. Place in the iodine solution for 2½-5 minutes.
  3. Rinse with distilled water, 3 times of 5-10 seconds each.
  4. Place in chloro-auric acid solution for 2½-5 minutes.
  5. Rinse with distilled water, 3 times of 5-10 seconds each.
  6. Place in fresh 3% hydrogen peroxide at 37°C for 6-36 hours.
  7. Dehydrate with ethanol, clear with xylene and mount using a resinous medium.

Expected Results

  • Amyloid – golden yellow to faint purple.
  • Erythrocytes, muscle and liver – blue
  • Nuclei and cytoplasm – brown
  • Collagen – grey

Safety Note

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


References

  1. Lynch, M.J. and Inwood, M.J.H., (1963),
    Gold as a permanent stain for amyloid,
    Stain Technology, v 38, page 260.
March 30, 2023
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