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Mayer’s Alum Hematoxylin Staining Variants

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

The solution usually meant when Mayer’s hemalum is specified is actually a modification of Mayer’s 1901 formula by Langeron. Mayer published several alum hematoxylin variants for nuclear staining, both progressive and regressive. Such solutions usually contain hematoxylin and an alum, and are called hemalum or alum hematoxylin solutions. Many formulae have been suggested. They vary in the amount of hematoxylin, the amount and type of aluminum salts, solvents, oxidizing agents and stabilizers.

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March 30, 2023
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Hale’s Colloidal Iron for Acid Mucosubstances

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

Hale's Colloidal Iron

for Acid Mucosubstances

10
steps
9
materials
print

Using the colloidal iron suspension of Rhinehart and Abu’l Haj.

Materials

Solutions

  • Neutral red
  • Acetic acid, 2M (12%)
  • Potassium ferrocyanide, 2% aqueous
  • Hydrochloric acid, 2% aqueous
  • Rhinehart & Abu’l Haj’s colloidal iron suspension

Working colloidal iron

MaterialAmount
Colloidal iron suspension1vol
Acetic acid, 2M1vol

Perls’ solution

MaterialAmount
2% potassium ferrocyanide1vol
2% hydrochloric acid1vol

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are usually satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into the working colloidal iron for 15-20 minutes.
  3. Wash with distilled water.
  4. Wash with running tap water for 5 minutes to remove all traces of colloidal iron
  5. Wash with distilled water.
  6. Place into freshly made Perls’ solution for 10 minutes.
  7. Wash with distilled water.
  8. Counterstain with neutral red for 1 minute.
  9. Dehydrate with ethanols.
  10. Clear with xylene and mount with a resinous medium.

Expected Results

  • Acid mucopolysaccharides  –  blue
  • Nuclei  –  red

Notes

  • The original method used a commercial colloidal iron preparation. This is still available. However, the colloidal iron suspension of Rhinehart and Abu’l Haj is reputed to produce a cleaner background. Other colloidal iron suspensions have also been recommended.
  • Since this method depends on the staining of iron compounds with the prussian blue reaction, any hemosiderin present will also be stained. If this is a concern, a control section should be stained which has not been treated with colloidal iron. Material stained blue in both sections should be discounted.
  • Nuclear fast red may also be used as a nuclear counterstain, or a Feulgen’s nucleal reaction may be applied before step 2, in which case the nuclear counterstain should be omitted.
  • A PAS may be applied following step 7, in which case the color of the nuclear counterstain should be changed, perhaps with a strictly progressive hemalum. Acid mucosubstances will be stained blue in contrast to red neutral mucosubstances. However, they are often present as mixtures and the contrast may not be clear.
  • Longley’s variant of this method includes a Feulgen’s nucleal reaction before step 2, and a Wiegert van Gieson counterstain following step 7, so that nuclei are black, cytoplasm is yellow and collagen red.

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.
  2. Bancroft, J.D. and Stevens A. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
  3. Lillie, R.D., (1954)
    Histopathologic technique and practical histochemistry Ed.2
    Blakiston, New York, USA.
March 30, 2023
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Hughesdon’s Metachromatic Stain

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

Hughesdon's Metachromatic Stain

8
steps
4
materials
print

Materials

Staining solution

MaterialAmount
Azure A0.2g
Distilled water100mL

Differentiating solution

MaterialAmount
Uranyl nitrate0.2g
Distilled water100mL

Tissue Sample

Paraffin sections of neutral buffered formalin fixed tissue are suitable. Mercuric chloride fixatives are reputed to emphasise metachromasia. Other fixatives may be satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Do a Mallory bleach using 1% potassium permangate alone for 5 minutes.
  3. Place in to staining solution for 5 minutes.
  4. Rinse with tap water.
  5. Place into the differentiating solution for 10 seconds.
  6. Rinse with tap water.
  7. Blot dry, dehydrate with absolute ethanol.
  8. Clear with xylene and mount with a synthetic resinous medium.

Expected Results

  • Acid mucopolysaccharides – Red/purple
  • Mast cells – Red/purple
  • Background – Blue

Notes

  • The time of differentiation should be adjusted to give good color contrast.
  • Metachromatically stained materials include acid mucins, cartilage, connective tissue ground substance, and mast cell granules.
  • It may be difficult to obtain uranyl nitrate due to restrictions placed on its distribution in some jurisdictions.

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. pp. 2250
    Oxford University Press, London, England.
March 30, 2023
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McNulty-Smith’s Zirconyl Hematoxylin

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

McNulty-Smith's Zirconyl Hematoxylin

6
steps
6
materials
print

This hematoxylin substitutes for alcian blue pH 2.5 for staining acid mucins.

Materials

IngredientAmountFunction
Hematoxylin100 mgDye
Ethanol, absolute5 mLSolvent
Sodium iodate, 0.5% aqu.5 mLOxidant
Zirconyl chloride, octahydrate400 mgMordant
Distilled Water22.5 mLSolvent
Glycerol7.5 mLAnti-oxidant

Compounding Procedure

  1. Dissolve the hematoxylin in the ethanol.
  2. Add the sodium iodate solution (freshly made).
  3. Add the zirconyl chloride.
  4. Combine the glycerol and water, and add to the solution.
  5. Stir for 5 minutes.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 10 minutes.
  3. Wash with distilled water, two changes of 2 minutes each.
  4. Counterstain in methylene green for 5 minutes.
  5. Wash with distilled water, two changes of 2 minutes each.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  green
  • Acid mucins  –  Purple

Notes

  • Formalin fixed, paraffin sections are suitable.
  • The counterstain recommended was 0.05% methylene green in 2.4% boric acid.
  • This method was recommended as a substitute for alcian blue staining of acid mucins (pH 2.5).
  • Staining may be removed with 1% hydrochloric acid in 70% ethanol.
  • The solution above is McNulty’s modification of Smith’s original, and stains more intensely:
    • Hematoxylin, 100 mg
    • Absolute ethanol, 5 mL
    • 0.1% aqueous sodium iodate, 5 mL
    • Zirconyl chloride octahydrate, 400 mg
    • 22% aqueous glycerol, 45 mL
    • Glacial acetic acid, 5 mL

Safety Note

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

References

  1. Smith, A. A., (1999)
    Zirconyl hematoxylin staining of acidic mucins.
    Journal of Histochemistry and Cytochemistry, v. 47, p. 1645.
  2. McNulty, J. M. and Smith, A. A., (2004)
    An improved formulation of the zirconyl hematoxylin stain for acidic mucins.
    Biotechnic and Histochemistry, v. 79, Nº 5, p. 191.
  3. McNulty, J. M., Kambour, M. J. and Smith, A. A., (2004)
    Use of an improved zirconyl hematoxylin stain in the diagnosis of Barrett’s esophagus.
    J. Cell. Mol. Med., v. 8, Nº 3, p. 382.
March 30, 2023
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Fluorescent Schiff

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

Fluorescent Schiff

11
steps
7
materials
print

This method demonstrates fungi, and may also be called a Fluorescent Gridley or CAFS.

Materials

  • Fluorescent Schiff reagent
  • Solution A
    MaterialAmount
    Chromium trioxide50g
    Distilled water500mL
  • Solution B
    MaterialAmount
    Sodium sulfite5g
    Distilled water500mL
  • Solution C
    MaterialAmount
    Ethanol, 70%495mL
    Hydrochloric acid5mL

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 10 minutes.
  3. Wash well with tap water.
  4. Bleach with solution B for 1 minute.
  5. Wash well with tap water.
  6. Rinse with distilled water.
  7. Place in fluorescent Schiff reagent for 20 minutes.
  8. Wash well with tap water.
  9. Place in solution C, two changes, 5 minutes each.
  10. Wash well with tap water.
  11. Dehydrate, clear and mount in a non fluorescent resinous medium.

Expected Results

Using a BG 12 exciter filter, and OG 4 (yellow) and/or OG5 (orange) barrier filter, fungi fluoresce yellow with acriflavine and green-red with acridine orange.

Notes

  • This is most useful as a screening method.
  • If background fluorescence is too bright for fungi to be distinguished,it may be quenched with an alum hematoxylin for 1 minute,or 0.5% aqueous potassium permanganate for 1 minute.Quench immediately before the final dehydration step.This should be done with caution as it may reduce fungal fluorescence.

Safety Note

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

References

No specific reference is given for this method. A similar technique using periodic acid as the oxidant is found in:

  1. Culling C.F.A., (1974)
    Handbook of histopathological and histochemical techniques Ed. 3
    Butterworth, London, UK.
March 30, 2023
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Masson 44/41 for Fibrin

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

Masson 44/41

for Fibrin

12
steps
12
materials
print

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 acidto saturation
    Mercuric chlorideto saturation
  • Plasma stain
    MaterialAmount
    Ponceau 6R1g
    Acetic acid, glacial1mL
    Distilled water99mL
  • Fibre stain
    MaterialAmount
    Naphthalene blue black CS1g
    Acetic acid, glacial1mL
    Distilled water99mL
  • Polyacid
    MaterialAmount
    Phosphotungstic acid1g
    Distilled water100mL

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 plasma stain for 5 minutes.
  8. Differentiate with the polyacid for 5 minutes.
  9. Place in the fibre stain for 30 minutes.
  10. Rinse briefly with 1% aqueous acetic acid.
  11. Dehydrate with ethanol.
  12. Clear with xylene and mount with a resinous medium.

Expected Results

  • Fresh fibrin  –  red
  • Older fibrin  –  black
  • Connective tissue  –  pale blue
  • Plasma cell inclusions  –  red

Notes

  • Ponceau 6R is also known as acid red 44.
  • Naphthalene blue black CS is also known as acid black 41.

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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Periodic Acid Fluorescent Schiff

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

Periodic Acid Fluorescent Schiff

9
steps
2
materials
print

Materials

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Protocol

  1. Dewax and hydrate sections.
  2. Place in periodic acid for 10 minutes.
  3. Wash well with tap water.
  4. Rinse with distilled water.
  5. Place in fluorescent Schiff reagent for 20 minutes.
  6. Wash well with tap water.
  7. Place in acid alcohol, two changes, 5 minutes each.
  8. Wash well with tap water.
  9. Dehydrate, clear and mount in a non fluorescent resinous medium.

Expected Results

Using a BG 12 exciter filter, and OG 4 (yellow) and/or OG5 (orange) barrier filter, 1-2-glycols fluoresce yellow with acriflavine and green-red with acridine orange.

Periodic acid fluorescent schiff

Notes

  • The same materials fluoresce as would be red or pink in a regular PAS reaction.
  • If background fluorescence is too bright it may be quenched with alum hematoxylin for 1 minute,or 0.5% aqueous potassium permanganate for 1 minute. Quench immediately before the final dehydration step.This should be done with caution as it may reduce fluorescence.

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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