Category

Stain Type

Held’s Molybdenum Hematoxylin

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

Held's Molybdenum Hematoxylin

6
steps
6
materials
print

Materials

Stock Solution

MaterialVariation IVariation IIFunction
Hematoxylin1 g1 gDye
Phosphomolybdic acid15 g–Mordant
Molybdic acid–excessMordant
Distilled water30 mL30 mLSolvent
95% ethanol70 mL70 mLSolvent

Solution A

MaterialAmountFunction
Ferric ammonium sulfate5 gMordant
Distilled water100 mLSolvent

Solution B

MaterialAmountFunction
Stock solution5 mL–
Distilled water95 mLSolvent

Compounding Procedures

Stock solution

  1. Mix the water and ethanol together.
  2. Dissolve the hematoxylin.
  3. Add the molybdenum compound specified and shake frequently over a period of one month until there is a distinct colour change to a deep blue-black, then decant.
  4. The staining improves with aging.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Optionally, place into solution A for 24 hours.
  3. Place into solution B for 12-24 hours.
  4. Differentiate with solution A if overstained.
  5. Wash in running tap water.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Tissue components  –  various shades.

Notes

  • Variation I is from the Microtomist’s Formulary and Guide and specifies phosphomolybdic acid, variation II is from the Microtomists Vade Mecum and specifies molybdic acid.
  • It is recommended for developing nerve tissues.

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:
    Bolles-Lee, A.; Edited by Gatenby, J. B. & Painter, T. S., (1937)
    The microtomists’s Vade-Mecum, p. 374
    Blakiston, Philadelphia, USA
  2. Bolles Lee, A.; Edited by Gatenby, J.B. and Beams, H.W., (1950)
    The Microtomist’s Vade-Mecum. 11 ed.,
    Churchill, London, UK.
March 30, 2023
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Slidders’ Fuchsin-Miller for Fibrin

By Fibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Yellowsolve Staining
Protocol

Slidders’ Fuchsin-Miller

for Fibrin

16
steps
7
materials
print

Materials

  • An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
  • Fuchsin
    MaterialAmount
    Acid fuchsin1g
    Acetic acid, glacial2.5mL
    Distilled water100mL
  • Miller
    MaterialAmount
    Milling yellow 3G2.5g
    2-ethoxy-ethanol100mL
  • Phosphotungstic acid
    MaterialAmount
    Phosphotungstic acid1g
    Distilled water100mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate or B5 overnight. Paraffin process overnight. Overnight formalin fixation and paraffin processing can produce acceptable results if sections are refixed for an hour in Bouin’s picro-acetic-formalin mixture at 56°C. Avoid rapid fixation with formalin and short processing, as this produces tissues that stain poorly even with the secondary fixation specified. Optimal results are obtained with extended mercuric chloride fixation, thorough processing, degreasing and secondary fixation of sections. Sections should be 3-5 µ thick.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. If mercury fixed, remove with the iodine-thiosulphate sequence.
  3. If not mercury fixed, place into Bouin’s fluid at 56°C for 1 hour.
  4. Rinse well with distilled water.
  5. Stain nuclei with an acid resistant nuclear stain.
  6. Rinse with 95% ethanol.
  7. Place in fuchsin for 10 minutes.
  8. Rinse with distilled water.
  9. Differentiate with phosphotungstic acid for 5 minutes.
  10. Rinse well with distilled water.
  11. Blot.
  12. Rinse well with cellosolve.
  13. Place into milling yellow until fibrin is red and tissues are yellow.
    This step takes from ½ – 4 hours.
  14. Rinse briefly with 1% aqueous acetic acid.
  15. Rinse with cellosolve.
  16. Clear with xylene and mount with a resinous medium.

Expected Results

  • Fibrin  –  red
  • Other tissue  –  yellow
  • Nuclei  –  black

Notes

  • It is very important that the milling yellow solution be completely anhydrous. Even small amounts of water or other solvents can cause problems with displacement and incomplete removal of red dye. For that reason, step 12 should be thorough and the milling yellow should be used in a Coplin jar with a lid sealed with tape.
  • A well stained section would show red fibrin only, muscle and erythrocytes being yellow. With poorly fixed material, both erythrocytes and muscle fibres may resist displacement of the red dye and they may have some residual red colouring. Sometimes this may be overcome by prestaining either with saturated picric acid in absolute ethanol or the MSB martius yellow solution for two minutes immediately before step 7, but a better resolution is correct fixation and processing.
  • Some intracellular materials may stain red, such as Paneth cell granules.

Safety Note

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

References

  1. Drury, R.A.B. and Wallington, E.A., (1980)
    Carleton’s histological technique Ed. 5
    Oxford University Press, Oxford, UK.
  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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Martius, Scarlet and Blue (MSB) for Fibrin

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

Martius, Scarlet and Blue (MSB)

for Fibrin

14
steps
8
materials
print

The MSB (Martius, Scarlet and Blue) method for fibrin is a reliable technique. It is more automatic than other methods, i.e. it is less dependent on skill and experience and is consequently suitable for a routine laboratory. Overnight mercuric chloride fixation (formol sublimate or B5) is preferred, followed by overnight paraffin processing, although formalin fixed, paraffin embedded material can produce acceptable results if sections are refixed for an hour in Bouin’s picro-acetic-formalin mixture at 56°C. Optimal results are obtained with extended mercuric chloride fixation, thorough processing, degreasing and secondary fixation of sections, as for the Picro-Mallory.

Materials

  • An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
  • Martius
    MaterialAmount
    Martius yellow0.5g
    Phosphotungstic acid2g
    Ethanol, 95%100mL
  • Scarlet
    MaterialAmount
    Crystal scarlet1g
    Acetic acid, glacial2.5mL
    Distilled water97.5mL
  • Blue
    MaterialAmount
    Methyl blue0.5g
    Acetic acid, glacial1mL
    Distilled water98mL
  • Phosphotungstic acid
    MaterialAmount
    Phosphotungstic acid1g
    Distilled water100mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate (or B5) overnight. Paraffin process overnight. Overnight formalin fixation is usually satisfactory, but avoid rapid fixation with formalin and short processing, as this produces tissues that stain poorly even with the secondary fixation specified. Sections should be 3-5 µ thick.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. If mercury fixed, remove with the iodine-thiosulphate sequence.
  3. If not mercury fixed, place into Bouin’s fluid at 56°C for 1 hour.
  4. Rinse well in distilled water.
  5. Stain nuclei with an acid resistant nuclear stain.
  6. Rinse with 95% ethanol.
  7. Place in martius yellow for 2 minutes.
  8. Rinse with distilled water.
  9. Place in crystal scarlet for 10 minutes.
  10. Differentiate with phosphotungstic acid until only fibrin is red (up to 10 minutes).
  11. Place in methyl blue until collagen is blue (up to 10 minutes).
  12. Rinse briefly with 1% aqueous acetic acid.
  13. Dehydrate rapidly with ethanol.
  14. Clear with xylene and mount with a resinous medium.

Expected Results

  • Fibrin  –  red
  • Fresh fibrin  –  yellow
  • Erythrocytes  –  yellow
  • Connective tissue  –  blue

Notes

  • Crystal scarlet is more commonly known as ponceau 6R.
  • Steps 10 & 11 specify the time as up to. These times should be established by inspection, but will generally remain consistent.
  • Lendrum’s recommendation for formalin fixed material was to dewax sections and treat with trichlorethylene in a sealed contained for 48 hours at 56°C, then to refix in absolute ethanol saturated with both picric acid and mercuric chloride for 24 hours before proceeding to step 2. The alternative treatment with Bouin’s fluid given at step 3 is often satisfactory.
  • Bancroft notes that dyes other than those originally given may be used. Some may not be easily available, and no CI numbers were given.
    ColorDye Options
    YellowLissamine fast yellow
    BlueDurazol blue
    Pontamine sky blue
    Fast green FCF
    Naphthalene black 10B
    RedPonceau de xylidine
    Azofuchsin

Safety Note

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

References

  1. Drury, R.A.B. and Wallington, E.A., (1980)
    Carleton’s histological technique Ed. 5
    Oxford University Press, Oxford, UK.
  2. Culling C.F.A., (1974)
    Handbook of histopathological and histochemical techniques Ed. 3
    Butterworth, London, UK.
  3. 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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Obadiah for Fibrin

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

Obadiah

for Fibrin

14
steps
11
materials
print

The name of this stain comes from the letters OBDR45, which refer to the dyes used: Orange, Blue, Direct Red 45 (a synonym for one of the red dyes).

With this trichrome stain full and complete fixation is absolutely essential. Minimalist formalin fixation and quick processing will give poorly stained erythrocytes with red or orange tinges instead of the yellow they should have. Lendrum and coworkers specified about seven days fixation in formal sublimate followed by processing thoroughly, sectioning, degreasing with trichlorethylene for 48 hours, then refixing in picro-mercuric-alcohol. Results are usually poor with formalin fixed material, even if treated with Bouin’s fluid at 56°C for an hour or so.

Materials

  • An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
  • Picro-mercuric ethanol
    MaterialAmount
    Ethanol, absolute100mL
    Picric acidtosaturation
    Mercuric chloridetosaturation
  • Orange
    MaterialAmount
    Orange G0.5g
    Phosphotungstic acid1g
    Ethanol, 95%100mL
  • Blue
    MaterialAmount
    Naphthalene blue black CS1g
    Acetic acid, glacial1mL
    Distilled water99mL
  • Red – Option 1
    MaterialAmount
    Chicago red2.5g
    Acetic acid, glacial2.5mL
    Distilled water97.5mL
  • Red – Option 2
    MaterialAmount
    Polar brilliant red BN1g
    Acetic acid, glacial1mL
    Distilled water99mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate for one week. They should be paraffin processed with a schedule that thoroughly and completely dehydrates, then thoroughly cleared with xylene and infiltrated with paraffin wax. This process would normally take 48 hours or longer. Err on the side of completeness, and do not attempt to shorten the process. Avoid rapid fixation and overnight processing, as this produces tissues that stain poorly. Sections should be 3-5 µ thick.

Protocol

  1. Dewax sections with xylene.
  2. Place into a sealed container of trichlorethylene at 56°C for 48 hours.
  3. Rinse well with absolute ethanol.
  4. Refix sections in picro-mercuric-ethanol for a minimum of 3 and preferably 24 hours.
  5. Remove mercury pigment with the iodine-thiosulphate sequence.
  6. Stain nuclei with an acid resistant nuclear stain.
  7. Place in the orange stain for 2 minutes.
  8. Rinse with distilled water.
  9. Place in the Blue stain up to 30 minutes.
  10. Differentiate with the polyacid for 5 minutes.
  11. Place in the Red stain 15-20 minutes (polar brilliant red) or 15-30 minutes (chicago red).
  12. Rinse with distilled water.
  13. Dehydrate with ethanol.
  14. Clear with xylene and mount with a resinous medium.

Expected Results

  • Old fibrin  –  black
  • Younger fibrin  –  may be yellow
  • Connective tissue  –  red

Notes

  • Note that this method reverses the usual order and uses a blue stain before the red stain.
  • Lendrum considered that this method demonstrated fibrin that other methods stained as collagen.

Safety Note

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

References

  1. Lendrum, A. C., et. al. (1962)
    Studies on the character and staining of fibrin.
    Journal of clinical pathology, v. 15, p. 401.
March 30, 2023
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Picro-Mallory for Fibrin – Long Version

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

Picro-Mallory

for Fibrin – Long Version

15
steps
12
materials
print

Lendrum, Fraser and others published several fibrin stains, the most well known being the Picro-Mallory. There are several variants of the method, some more complicated than others.

See this alternative protocol for a shorter version.

With this trichrome stain full and complete fixation is absolutely essential. Minimalist formalin fixation and quick processing will give poorly stained erythrocytes with red or orange tinges instead of the yellow they should have. Lendrum and coworkers specified about seven days fixation in formal sublimate followed by processing thoroughly, sectioning, degreasing with trichlorethylene for 48 hours, then refixing in picro-mercuric-alcohol. Adequate results can be obtained with thorough formalin fixation (24 hours), thorough processing (overnight), sectioning, followed by refixing in Bouin’s fluid at 56°C for an hour or so. However, results are inferior to those obtained by the full procedure. It is also a method where experience is required for optimal results.

Materials

  • An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the
    celestine blue-hemalum sequence.
  • Yellow mordant
    MaterialAmount
    Picric acid, sat. in 80% ethanol100mL
    Orange G0.2g
    Lissamine yellow0.2g
  • Stock differentiator
    MaterialAmount
    Picric acid2.5g
    Ethanol, 95%100mL
    Phosphotungstic acid25g

    Dissolve ingredients and filter.

  • Red differentiator
    MaterialAmount
    Stock differentiator40mL
    Ethanol, 95%20mL
    Distilled water90mL
  • Blue differentiator
    MaterialAmount
    Stock differentiator10mL
    Distilled water90mL
  • Red stain
    MaterialOption 1Option 2Option 3
    Acid fuchsin1g–0.4g
    Lissamine fast red––0.2g
    Biebrich scarlet–1g–
    Acetic acid, glacial1mL1mL1mL
    Distilled water99mL99mL99mL
  • Blue stain
    MaterialAmount
    Methyl blue1g
    Acetic acid, glacial1mL
    Distilled water99mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate for one week. They should be paraffin processed with a schedule that thoroughly and completely dehydrates, then thoroughly cleared with xylene and infiltrated with paraffin wax. This process would normally take 48 hours or longer. Err on the side of completeness, and do not attempt to shorten the process. Avoid rapid fixation and overnight processing, especially with formalin fixatives, as this produces tissues that stain poorly even with secondary fixation such as Bouin’s fluid at 56°C for an hour. Sections should be 3-5 µ thick.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Remove mercury pigment with the iodine-thiosulphate sequence.
  3. Stain nuclei with an acid resistant nuclear stain.
  4. Place in yellow mordant for 2-3 minutes.
  5. Wash in distilled water until only erythrocytes are yellow.
  6. Place in the red stain for 5-10 minutes.
  7. Rinse with 1% aqueous acetic acid.
  8. Differentiate with the red differentiator until fibrin is prominent microscopically.
  9. Rinse well in distilled water.
  10. Place in the blue stain for 5 minutes.
  11. Rinse briefly with 1% aqueous acetic acid.
  12. Place in the blue differentiator for 1-2 minutes.
  13. Biefly rinse with 1% aqueous acetic acid.
  14. Dehydrate with ethanol.
  15. Clear with xylene and mount with a resinous medium.

Expected Results

  • Fibrin  –  clear red
  • Erythrocytes  –  yellow
  • Connective tissue  –  blue

Notes

  • The most common dye combination is acid fuchsin and methyl blue.
  • Adequate results may be obtained with 24-48 hours fixation in neutral buffered formalin, overnight paraffin processing and secondary fixation of sections with Bouin’s picro-formol-acetic for 1 hour at 56°C.
  • Good results are obtained with the fixation and processing recommended in the text.
  • Optimal results are obtained with the fixation and processing recommended in the text, followed by degreasing and secondary fixation in picro-mercuric ethanol.

    Replace step 1 with the following:

    1. Dewax sections with xylene.
    2. Place in trichlorethylene in a sealed container at 56°C for 48 hours.
    3. Rinse sections well with absolute ethanol.
    4. Place in picro-mercuric-ethanol for 24 hours.
    5. Wash sections with water, and continue from step 2.

Safety Note

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

References

  1. Lendrum, A. C., et. al. (1962)
    Studies on the character and staining of fibrin.
    Journal of clinical pathology, v. 15, p. 401.
March 30, 2023
Love0

Picro-Mallory for Fibrin – Shorter Version

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

Picro-Mallory

for Fibrin – Shorter Version

17
steps
10
materials
print

This version of the picro-Mallory is relatively uncomplicated and is suitable for a routine pathology laboratory. Proper fixation is still essential and minimalist formalin fixation with quick processing should be avoided as it will give disappointing results with poorly stained erythrocytes. Originally, extended mercury fixation, thorough processing, degreasing and refixing in picro-mercuric-ethanol were specified. Although fixation in formol sublimate (or B5) is preferred for this method as well, adequate results can be obtained with overnight formalin fixation and overnight processing. Refixation of sections in Bouin’s fluid at 56°C for an hour or so overcomes some of the deficiencies in fixation, but results are still inferior to those obtained by the full procedure.

Materials

  • An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
  • Picro-orange
    MaterialAmount
    Picric acid, sat. in 80% ethanol100mL
    Orange G0.2g
  • Yellow differentiator
    MaterialAmount
    Picro-orange30mL
    Ethanol, 80%70mL
  • Red stain
    MaterialAmount
    Acid fuchsin1g
    Acetic acid, glacial1mL
    Distilled water99mL
  • Red differentiator
    MaterialAmount
    Phosphotungstic acid1g
    Distilled water100mL
  • Blue stain
    MaterialAmount
    Methyl blue2g
    Acetic acid, glacial2mL
    Distilled water98mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate (or B5) overnight. Paraffin process overnight. Avoid rapid fixation with formalin and short processing, as this produces tissues that stain poorly even with the secondary fixation specified. Sections should be 3-5 µ thick.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Remove mercury pigment with the iodine-thiosulphate sequence.
  3. Place into Bouin’s fluid at 56°C for 1 hour.
  4. Rinse well in distilled water.
  5. Stain nuclei with an acid resistant nuclear stain.
  6. Rinse with 95% ethanol.
  7. Place in picro-orange for 2 minutes.
  8. Without rinsing, place in the red stain for 5 minutes.
  9. Rinse with distilled water.
  10. Dip into yellow differentiator.
  11. Rinse in distilled water.
  12. Differentiate with the red differentiator for 5 minutes.
  13. Rinse with distilled water.
  14. Place in the blue stain for 2 minutes.
  15. Rinse briefly with 1% aqueous acetic acid.
  16. Dehydrate with ethanol.
  17. Clear with xylene and mount with a resinous medium.

Expected Results

  • Fibrin  –  clear red
  • Fibrinoid  –  varies from red to orange
  • Erythrocytes  –  orange
  • Connective tissue  –  blue

Notes

  • If possible, the extended fixation and slow processing specified for the longer version should be used. Degreasing and refixation will also improve results if time allows.

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., (1974)
    Handbook of histopathological and histochemical techniques Ed. 3
    Butterworth, London, UK.
March 30, 2023
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Yellowsolve I for Fibrin

By Fibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Yellowsolve Staining
Protocol

Yellowsolve I

for Fibrin

13
steps
9
materials
print

Materials

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are likely suitable. It should be noted, however, that the authors of this method favoured extended fixation in formal sublimate of up to 10 days for fibrin. This fixative is now deprecated due to its mercuric chloride content. With other staining methods, pretreatment of sections with Bouin’s fluid for an hour at 60°C can compensate for the lack of mercury fixation to a large degree.

Protocol

  1. Dewax sections with xylene.
  2. Place in trichlorethylene for 24 to 48 hours.
  3. Bring sections to water.
  4. Stain elastic fibres if desired.
  5. Stain nuclei with the celestine blue-hemalum sequence.
  6. Wash well with water.
  7. Place in solution A for 30 minutes.
  8. Rinse with 2-ethoxyethanol then with tetrachlorethylene.
  9. Place into tetrachlorethylene overnight.
  10. Rinse with 2-ethoxyethanol.
  11. Differentiate with solution B, controlling microscopically.
  12. Rinse with 2-ethoxyethanol.
  13. Clear in xylene and mount with a synthetic resinous medium.

Expected Results

  • Fibrin  –  red
  • Background  –  yellow
  • Nuclei  –  black
  • Acidophil cytoplasmic inclusions  –  red

Notes

  • 2-Ethoxyethanol is also known as cellosolve or ethylene glycol monoethyl ether, and has the formula CH3CH2OCH2CH2OH.
  • Tetrachlorethylene has the formula Cl2C=CCl2 or C2Cl4
  • Trichlorethylene has the formula ClHC=CCl2 or C2HCl3
  • Both trichlorethylene and tetrachlorethylene should be used in a fume hood.

Safety Note

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

References

  1. Lendrum A C, Fraser D S, Slidders W and Henderson R. (1962)
    Studies on the character and staining of fibrin.
    Journal of clinical pathology, v. 15, p. 401.
March 30, 2023
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Shum & Hon’s PTAH

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

Shum & Hon's PTAH

6
steps
3
materials
print

Materials

MaterialAmountFunction
Hematein,0.8 g.Dye
Phosphotungstic acid9 gMordant
Distilled water1 LSolvent

Compounding procedures

  1. Combine the hematein, phosphotungstic acid and 10 mL of the water and grind them to a paste with a pestle and mortar.
  2. Wash the contents into a beaker with the rest of the water.
  3. Bring the solution to a boil, then cool and filter.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Perform a Mallory bleach using 0.25% potassium permanganate.
  3. Rinse well with water.
  4. Place into the PTAH solution for 12 – 24 hours at room temperature.
  5. Rinse well with water.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei, erythrocytes, fresh fibrin, muscle striations  –  blue
  • Background  –  red

Notes

  • Observe the colour of the sludge while grinding as a good sample of hematein will be chocolate brown. If the sludge is pale, the final solution may not be satisfactory.
  • The staining may be done in an oven at about 60°C for a few hours, but the results are often less satisfactory.
  • Many substances have been stated to be stained blue. Sometimes the red staining overshadows blue staining, and the section may need to be washed to remove excess red.

Safety Note

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

References

  1. Histological demonstration techniques, (1974)
    Cook, H C.
    Butterworths, London, England.
March 30, 2023
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Foot’s Impregnation for Reticulin

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

Foot's Impregnation

for Reticulin

22
steps
22
materials
print

Materials

MaterialVariation IVariation IIVariation IIIVariation IV
Lugol’s iodine++++
Sodium thiosulphate, 1% aqu.++++
Sodium thiosulphate, 5% aqu.++++
Potassium permanganate, 0.25% aqu.++++
Oxalic acid, 5% aqu.++++
Strong ammonium hydroxide (s.g. 0.88)++++
Ammonium hydroxide, 0.1% aqu–+––
Foot’s tannin sol.–+––
Silver nitrate, aqu.2% & 10%1% & 10%10%10%
Formalin, aqu.5%20%20%1%
Yellow gold chloride, aqu.1%–0.2%0.2%
Foot’s gold sublimate–+––
Alkali, aqu.NaOH 40%KOH 40%LiC03 sat.Na2CO3
Weigert’s 1903 iron hematoxylin+–––
Harris alum hematoxylin––+–
van Gieson’s picro-fuchsin+–+–
Neutral red, 1% aqu.–––+

Lugol’s Iodine (see note)

MaterialAmount
Iodine6g
Potassium iodide4g
Distilled Water100mL

Mix the iodine and potassium iodide in a 200 mL flask. Add 10 mL of the water. When the iodine has dissolved make up to 100 mL with distilled water.

Foot’s gold sublimate

MaterialAmount
Yellow gold chloride0.2g
Mercuric chloride0.5g
Distilled Water100mL

Foot’s tannin solution

MaterialAmount
Tannic acid0.15g
Ammonium bromide3.5g
Strong formalin5mL
Distilled Water100mL

Preparation of Foot’s Ammoniacal Silver

Variation I

  1. Place 25 mL of 10% silver nitrate in a flask.
  2. Add 1 mL of 40% sodium hydroxide.
  3. Add ammonium hydroxide by drops until the precipitate is almost dissolved.
  4. Dilute to 100 mL with distilled water.

Variation II

  1. Place 10 mL of 1% silver nitrate in a flask.
  2. Add 0.1 mL of 40% potassium hydroxide.
  3. Add ammonium hydroxide by drops until the precipitate is almost dissolved.
  4. Dilute to 100 mL with distilled water.

Variation III

  1. Place 10 mL of 10% silver nitrate in a flask.
  2. Add 10 mL of saturated aqueous lithium carbonate.
  3. Let the precipitate settle, then decant the supernatent.
  4. Wash, allow to settle, and decant several times.
  5. Add 25 mL distilled water to the precipitate.
  6. Add ammonium hydroxide by drops until the precipitate is almost dissolved.
  7. Dilute to 100 mL with distilled water and filter.

Variation IV

  1. Place 10 mL of 10% silver nitrate in a flask.
  2. Add 40 mL of aqueous sodium carbonate (see note for concentration).
  3. Let the precipitate settle, then decant the supernatent.
  4. Wash, allow to settle and decant several times.
  5. Add ammonium hydroxide by drops until the precipitate is almost dissolved.

Tissue Sample

5 µ paraffin sections of Zenker fixed tissue are suitable. Other fixatives may be satisfactory. Carleton & Leach say that Var IV is suitable for 10% formalin, Zenker, Bouin or Helly fixed tissue. In all cases a section adhesive is recommended.

Protocol

StepVariation IVariation IIVariation IIIVariation IV
1Bring sections to water via xylene and ethanol (all variations).
2Place in Lugol’s iodine.5 min5 min5 min5 min
3Bleach in 1% sodium thiosulphate.30 sec30 sec30 sec30 sec
4Oxidise in 0.25% potassium permanganate.5 min5 min5 min5 min
5Bleach with 5% oxalic acid.10 min10 min10 min10 min
6Wash well with tap water.5 min5 min5 min5 min
7Rinse with distilled water.5 sec5 sec5 sec5 sec
8Place in 2% silver nitrate.48 hrs———
Place in Foot’s tannin solution at 37°C.—15 min——
Place in 0.1% ammonium hydroxide.—30 sec——
9Rinse with distilled water.5 sec5 sec——
10Place in Foot’s ammoniacal silver solution (Variation I).30 min———
Place in Foot’s ammoniacal silver solution (Variation II), 2 changes, each for:—5 min——
Place in Foot’s ammoniacal silver solution (Variation III) at 45°C.——15 min—
Place in Foot’s ammoniacal silver solution (Variation IV) at 45°C.———15 min
11Rinse with distilled water.5 sec5 sec5 sec—
12Reduce with 10% formalin.30 min———
Reduce with 20% formalin.—3 min2 min—
Flood with 1% formalin twice.———10 sec
13Rinse with distilled water.——5 sec—
Wash off with tap water.———5 sec
14Tone with 1% yellow gold chloride.1 hr———
Tone with Foot’s gold sublimate.3 min———
Tone with 0.2% yellow gold chloride.——2 min2 min
15Wash with distilled water.1 min1 min——
Wash with tap water.———2 min
16Place in 5% sodium thiosulphate.—3 min2 min2 min
17Wash with tap water.5 min5 min5 min5 min
18Place in Weigert’s iron hematoxylin.1 min———
Place in Harris’ alum hematoxylin.——3 min—
Place in neutral red.———1 min
19Wash with tap wate.5 min5 min5 min1 min
20Place in van Gieson’s picro-fuchsin.30 sec—45 sec—
22Dehydrate with ethanol, clear with xylene and mount in a resinous medium (all variations).

Expected Results

  • Reticulin fibres  –  black
  • Nuclei  –  as counterstained
  • Background  –  grey

Notes

  • Ensure that the ammonium hydroxide is fresh and full strength. Keep well stoppered when not in use. After removing the amount required, immediately restopper the bottle.
  • Improperly made ammoniacal silver solutions can affect the quality of the impregnation. There should be a faint, persistent opalescence, with only a faint smell of ammonia.
  • The formula for Lugol’s iodine may be an error. The amounts may have been reversed,
    i.e. It may be 4 g iodine and 6 g potassium iodide. Please refer to the page for
    Lugol’s iodine and compare Gray and Lee. The details for these methods are from Gray.
  • The instructions for ammoniacal silver solution Var IV says to add 40 mL of sodium carbonate to the silver nitrate solution. The sodium carbonate should be made as a 5% solution of the anhydrous salt or as an 11% solution of the dodecahydrate (12H2O).
  • 1%, 10% and 20% formalin solutions are made by diluting strong formalin appropriately with water, i.e.:
    • 1% is 1 mL strong formalin plus 99 mL water,
    • 10% is 10 mL strong formalin plus 90 mL water, and
    • 20% is 20 mL strong formalin plus 80 mL water.

Safety Note

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

References

  1. Gray, Peter. (1954), (for Var I, II & III)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
  2. Carleton, H M, and Leach, E H, (1938), (for Var IV)
    Histological technique., Ed. 2.
    Oxford University Press, London, England.
March 30, 2023
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Friedlander’s Alum Hematoxylin

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

Friedlander's Alum Hematoxylin

8
steps
5
materials
print

Materials

MaterialAmountFunction
Hematoxylin6 gDye
Potassium alum6 gMordant
Distilled water300 mLSolvent
95% ethanol300 mLSolvent
Glycerol300 mLStabiliser

Compounding procedures

  1. Dissolve the Alum in the water.
  2. Dissolve the hematoxylin in the ethanol.
  3. Combine, and 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

  • This is probably a regressive formula.
  • The 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.
March 30, 2023
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