Category

Stain Type

Gomori’s Aldehyde Fuchsin

By Aldehyde Fuchsin, Dye Type, Elastic Fibers, Intracytoplasmic Granules, Mast Cells, Protocols, Stain Target, Stain Type
Protocol

Gomori's Aldehyde Fuchsin

7
steps
4
materials
print

Materials

Solution A

MaterialAmount
Basic fuchsin1g
Paraldehyde, fresh1mL
Hydrochloric acid, conc.1mL
Ethanol, 70%200mL

Compounding Procedure

  1. Dissolve the dye in the ethanol.
  2. Add paraldehyde and hydrochloric acid.
  3. Ripen at room temperature for 48-72 hours.
  4. Refrigerate. The solution is stable for 2-3 months.

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. Wash with water.
  3. Rinse with 70% ethanol.
  4. Place in the staining solution for 10 minutes.
  5. Rinse well with 95% ethanol.
  6. Counterstain the nuclei and/or the cytoplasm if wished.
  7. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  purple
  • Mast cells  –  purple
  • Pituitary β cells  –  purple
  • Sulphated mucins  –  purple
  • Background  –  as the counterstain
  • Nuclei  –  as the nuclear stain

Notes

  • The basic fuchsin used for this solution should be one that is suitable for Schiff’s reagent, i.e., it should have a high pararosanilin content. Both methods involve forming a compound between an aldehyde and dye.
  • Light counterstaining with a progressive alum hematoxylin and eosin is also suitable.
  • Many other counterstains can be used, including methods such as Masson’s trichrome.
  • Gabe described a technique for the preparation and use of aldehyde fuchsin powder.

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.
March 30, 2023
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Lillie’s Allochrome

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

Lillie's Allochrome

8
steps
5
materials
print

Materials

  • 1% Aqueous periodic acid
  • Schiff’s reagent
  • Weigert’s iron hematoxylin
  • Picro-methyl blue
    MaterialAmount
    Saturated aqueous picric acid100mL
    1% aqueous methyl blue0.4mL

Tissue Sample

5 µ paraffin sections. Most fixatives are satisfactory, including 10% NBF, but those generally considered preferable for trichrome stains will be advantageous.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Do a PAS stain, but do not counterstain.
  3. Stain nuclei with Weigert’s iron hematoxylin or equivalent.
  4. Wash well in tap water, rinse with distilled water.
  5. Place into picro-methyl blue for 6 minutes.
  6. Rinse well with 95% ethanol.
  7. Dehydrate with absolute ethanol.
  8. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei – Brown-black
  • Collagen & reticulin – blue
  • Cytoplasm & muscle – yellow
  • PAS positive structures – 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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Bennhold’s Congo Red for Amyloid

By Amyloid, Direct Dye Staining, Protocols, Stain Target, Stain Type
Protocol

Bennhold's Congo Red

for Amyloid

8
steps
5
materials
print

Materials

  • Mayer’s hemalum
  • Congo red
    MaterialAmount
    Congo red1g
    Distilled water100mL
  • Lithium carbonate
    MaterialAmount
    Lithium carbonateasrequired
    Distilled water100mL

Dissolve to saturation.

Tissue Sample

Paraffin sections of formalin fixed tissues are satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into the congo red solution for
    1. 30 minutes at room temperature, or
    2. 45 minutes at 56°C, or
    3. 15 seconds at boiling.
  3. Drain and, without rinsing, place into lithium carbonate solution for 15 seconds.
  4. Drain and, without rinsing, differentiate in 80% ethanol. This usually takes just a few seconds.
  5. Wash well with tap water.
  6. Stain nuclei with hematoxylin and blue.
  7. Dehydrate with ethanol.
  8. Clear with xylene and mount with a resinous medium.

Expected Results

  • Amyloid  –  red
  • Nuclei  –  blue

Notes

  • Amyloid displays apple green birefringence when viewed with crossed polarisers, one above and one below the section.
  • The congo red is most commonly applied for 30 minutes at room temperature.
  • Differentiation in 80% ethanol is difficult to control, and amyloid is often poorly demonstrated. Due to this the method is not usually recommended.

Safety Note

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

References

  1. Bennhold, H., (1922),
    Eine specifische amyloidfärbung mit kongorot,
    Münchener Medizinische Wochenschrift, v 44, page 1537
March 30, 2023
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Bencosme’s HPS General Oversight stain

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

Bencosme's HPS

General Oversight stain

11
steps
5
materials
print

Also known as the Hematoxylin Phloxine Saffron stain.

Materials

  • Regressive hemalum
  • Solution A
    MaterialAmount
    Phloxine B2g
    Distilled water100mL

    Dissolve the phloxine B and filter. Preserve with 2-5 drops of strong formalin.

  • Solution B
    MaterialAmount
    Saffron6g
    Absolute ethanol200mL

    Mix the saffron stigmata with the ethanol and seal tightly. Place in an oven at 56°C for 1-2 weeks.

Tissue Sample

Brasil’s fixative was specified. 5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable, but results may be improved by refixing in Bouin’s fixative or saturated aqueous picric acid at 56°C for one hour prior to staining, then washing the sections in tap water to remove all yellow discoloration.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Treat with Bouin’s fluid if desired.
  3. Stain nuclei with hemalum, differentiate and blue.
  4. Wash well with water.
  5. Place in solution A for 2-20 minutes.
  6. Wash with tap water until red stain stops being removed, 1-5 minutes.
  7. Differentiate phloxine with 80% ethanol up to 1½ minutes.
  8. Thoroughly dehydrate with absolute ethanol.
  9. Place into solution B for 2-20 minutes until a suitable red-yellow balance is achieved.
  10. Rinse well with absolute ethanol.
  11. Clear with xylene and mount with a synthetic resinous medium.

Expected Results

  • Nuclei  –  blue
  • Cytoplasm  –  red shades
  • Muscle  –  pink
  • Elastic fibres  –  bright red
  • Collagen  –  orange-yellow

Notes

  • Any hemalum may be used, but Bencosme specified a particular formula.
  • Saffron is expensive. It may be available in East Indian grocery stores or health food stores as its most common use today is as a spice and food coloring. Usually, whole stigmata are more effective than ground saffron.
  • If the saffron solution is too strong it may be diluted with absolute ethanol. Store and use at room temperature. It has a limited life of about a month and is best when freshly made. It is important that there be no water in the saffron solution and that sections be thoroughly dehydrated before it is applied. If contaminated with moisture the solution must be discarded.

Safety Note

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

References

  1. Medical Laboratory Technology, 2nd ed. (1969)
    Lynch M.J., Raphael S.S., Mellor L.D., Spare P.D. and Inwood M.J.H.,
    W. B. Saunders Co., Toronto, On., Canada
March 30, 2023
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Congo Red Fluorescence for Amyloid

By Amyloid, Direct Dye Staining, Protocols, Stain Target, Stain Type

Protocol

Congo Red Fluorescence

for Amyloid

8
steps
4
materials
print

Expected Results

Congo red staining showing amyloid and lipofuscin in an autopsy specimen of senile cardiac amyloidosis

fig1-congo-red-staining-amyloid-lipofuscin-autopsy-senile-cardiac-amyloidosis-600x400

Figure 1. Congo Red Staining Showing Amyloid and Lipofuscin in an Autopsy Specimen of Senile Cardiac Amyloidosis

Congo red staining of an autopsy specimen of senile cardiac amyloidosis, showing amyloid (extracellular washed-out red material) and abundant lipofuscin (yellow granular material). Cardiac amyloidosis very high mag by Nephron / Michael Bonert, Pathology & Molecular Medicine, McMaster University is licensed under CC BY-SA 3.0.

  • Amyloid  –  Orange to red fluorescence

Materials

Congo red

MaterialAmount
Congo red0.1g
Ethanol, 50%100mL

Differentiator

MaterialAmount
Potassium hydroxide0.2g
Ethanol, 80%100mL

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 congo red for 1 minute.
  3. Wash with tap water.
  4. Place into differentiator until the section appears to be unstained.
  5. Wash with water.
  6. Dehydrate with absolute ethanol.
  7. Clear with xylene.
  8. Mount with a fluorescence free resinous medium.

Notes

  • This method is very similar to Highmans procedure.

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. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
March 30, 2023
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Eastwood and Cole Congo Red for Amyloid

By Amyloid, Direct Dye Staining, Protocols, Stain Target, Stain Type
Protocol

Eastwood and Cole Congo Red

for Amyloid

6
steps
7
materials
print

Materials

  • Mayer’s hemalum
  • Buffer pH 10
    MaterialAmount
    Glycine, 0.1M30mL
    Sodium chloride, 0.1M30mL
    Sodium hydroxide, 0.1M40mL
  • Congo red
    MaterialAmount
    Congo red0.5g
    Buffer pH 1050mL
    Ethanol, absolute50mL

    Combine the ethanol and buffer. Dissolve the congo red.

Tissue Sample

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

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with hemalum and blue.
  3. Place in congo red solution for 10 – 20 minutes.
  4. Rinse off the staining solution with 70% ethanol until clear.
  5. Dehydrate with ethanol.
  6. Clear with xylene and mount with a resinous medium.

Expected Results

  • Amyloid  –  Orange to red
  • Eosinophils and elastic  –  Orange to red
  • Nuclei  –  Blue

Notes

  • Amyloid displays apple green birefringence when viewed with crossed polarisers, one above and one below the section.

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. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
March 30, 2023
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Highman’s Congo Red for Amyloid

By Amyloid, Direct Dye Staining, Protocols, Stain Target, Stain Type
Protocol

Highman's Congo Red

for Amyloid

7
steps
6
materials
print

Materials

  • Mayer’s hemalum
  • Congo red
    MaterialAmount
    Congo red0.5g
    Distilled water50mL
    Ethanol, 100%50mL
  • Alkaline ethanol
    MaterialAmount
    Ethanol, 80%100mL
    Potassium hydroxide0.2g

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 into congo red solution for 5 minutes or longer.
  3. Differentiate in alkaline ethanol (about 5-30 seconds).
  4. Wash well with tap water.
  5. Stain nuclei with hematoxylin and blue.
  6. Dehydrate with ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Amyloid  –  orange red
  • Nuclei  –  blue
  • Background  – colorless

Notes

  • Amyloid displays apple green birefringence when viewed with crossed polarisers, one above and one below the section.
  • Sirius red F3B may also be used in this method.

Safety Note

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

References

  1. Highman, B., (1946),
    Improved methods for demonstrating amyloid in paraffin sections,
    Archives of Pathology, v 41, page 559
  2. Bancroft, J. D. and Stevens, A.
    Theory and practice of histological techniques,
    Churchill Livingstone, London, England
March 30, 2023
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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.

Read More
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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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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