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Mast Cells

Maximow’s Metachromatic Stain

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

Maximow's Metachromatic Stain

6
steps
2
materials
print

Materials

  • Ethanol, 50%, saturated with thionin.
  • Sodium carbonate, 0.3% w/v aqueous.

Tissue Sample

Alcohol fixed tissues are recommended, but5µ paraffin sections of neutral buffered formalin fixed tissue may be suitable. Other fixatives may be satisfactory.

Protocol

  1. Dewax with xylene and bring sections to 70% ethanol.
  2. Stain with the thionin solution for 24 to 48 hours.
  3. Blot section dry.
  4. Rinse twice with 95% ethanol.
  5. Rinse well with absolute ethanol.
  6. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei – blue
  • Mast cell granules – red/purple

Safety Note

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

References

  1. McManus, J.F.A. and Mowry, R.W., (1960),
    Staining methods, histologic and histochemical, pp. 261.
    Harper & Row, New York, NY, USA.
March 30, 2023
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Leder Esterase for Mast Cells

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

Leder Esterase

for Mast Cells

7
steps
12
materials
print

Materials

Pararosanilin

MaterialAmount
Pararosanilin0.5g
Distilled water20mL
Hydrochloric acid, conc.2.5mL

Warm gently and filter. Refrigerate.

Nitrosylated pararosanilin

MaterialAmount
Pararosanilin solution0.1mL
Sodium nitrite, 4% aqueous0.1mL

The sodium nitrite solution must be fresh. After mixing, let stand for one minute. Use immediately.

Sorenson Stock A

MaterialAmount
Disodium hydrogen phosphate2.73g
Distilled water250mL

Sorenson Stock B

MaterialAmount
Potassium dihydrogen phosphate2.27g
Distilled water250mL

Sorenson working buffer

MaterialAmount
Sorenson stock A41mL
Sorenson stock B9mL

Incubating medium

MaterialAmount
Naphthol-ASD chloroacetate10mg
N,N-dimethyformamide1mL
Sorenson’s working buffer35mL
Nitrosylated pararosanilin0.2mL

Combine in the order given. Mix well, filter and use immediately.

Light green

MaterialAmount
Light green SF yellowish1g
Distilled water100mL
Glacial acetic acid1mL

Tissue Sample

4µ paraffin sections of formalin fixed tissue are suitable.

Protocol

  1. Bring sections to distilled water.
  2. Place in the incubating medium for 40 minutes.
  3. Wash in running tap water for 5 minutes.
  4. Counterstain with light green for 30 seconds.
  5. Wash in running tap water for 5 min.
  6. Air dry.
  7. Clear in xylol and mount.

Expected Results

  • Esterase activity (mast cells)  –  red
  • Background  –  green

Notes

  • An alternative to air drying, which avoids some of the artifact introduced, is to blot the section, then wash with xylene. This is repeated until the sections are cleared.

Safety Note

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

References

  1. Leder, L.D. (1964)
    Klinische Wochenschrift
March 30, 2023
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Csaba’s Stain for Mast Cells

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

Csaba's Stain

for Mast Cells

5
steps
6
materials
print

Materials

Staining solution

MaterialAmount
Alcian blue0.36g
Safranin0.18g
Ferric ammonium sulfate0.48g
Walpole’s acetate HCl buffer100mL

Walpole’s acetate-HCl buffer, pH 1.42

MaterialAmount
Sodium acetate, M/1100mL
Hydrochloric acid, M/1120mL

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. Place in to staining solution for 10-20 minutes.
  3. Rinse with water.
  4. Dehydrate with tertiary butanol.
  5. clear with xylene and mount using a resinous medium.

Expected Results

  • Young mast cells – blue
  • Mature mast cells – red

Notes

  • To make an M/1 solution of sodium acetate, dissolve 82.04 grams anhydrous or 136.09 crystal sodium acetate in one litre of distilled water.
  • To make an M/1 solution of hydrochloric acid, dilute concentrated hydrochloric acid, which is about 10M, with distilled water (one part acid to 8 parts water) and titrate against an alkali of known molarity. Adjust the concentration to M/1 with distilled water.

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. pp. 169 & 419
    Butterworths, London, England.
March 30, 2023
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Simple Metachromatic Stain

By Intracytoplasmic Granules, Mast Cells, Protocols, Stain Target

Protocol

Simple Metachromatic Stain

6
steps
3
materials
print

Materials

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. Place in to staining solution for 5 minutes.
  3. Rinse well with tap water.
  4. If staining is too dark, differentiate with acetic acid until metachromasia is evident.
  5. Rinse well with tap water.
  6. Coverslip with water and examine wet, or blot dry and immerse in xylene. Repeat until cleared. Coverslip using a resinous medium

Expected Results

Dye colorBlueRedBrown
NucleiBlueRedBrown
Acid mucopolysaccharidesRed/purpleYellowYellow
BackgroundBlueRedBrown

Notes

  • The actual dye concentration is not too important. It should be strong enough to stain within 5 minutes. Concentrations between 0.1% and 1% are usually suitable.
  • The staining time can be varied. The staining solution should be applied for long enough to give dark staining. Usually this is about 2-5 minutes.
  • Methylene blue may be polychromed by making a 1% w/v solution and leaving it for several months in an airy, bright location with a loose stopper of cotton wool. When it gives good metachromatic staining, stopper tightly and place in a cupboard.
  • The acetic acid concentration may be varied. Usually a concentration of 0.1% to 1% is suitable. The higher the concentration, the faster dye is removed.
  • Sections may require little or no differentiation. Always check the staining before applying acetic acid. Stop when a distinct contrast in color is seen.
  • To mount in a resinous medium, blot the wet section dry and immerse in xylene. Repeat until the section is cleared and becomes transparent. Mount using a resinous medium.
  • Ethanol should be avoided as it may destroy any metachromasia.
  • Metachromatically stained materials include intestinal and other mucins, cartilage, connective tissue ground substance and mast cell granules.

Safety Note

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

March 30, 2023
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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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Aldehyde Toluidine Blue for Mast Cells

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

Aldehyde Toluidine Blue

for Mast Cells

7
steps
6
materials
print

Materials

Compounding Procedure

  1. Dissolve the dye in the ethanol.
  2. Add paraldehyde and acid.
  3. Ripen one week at room temperature.
  4. Store at room temperature.
  5. Filter before use. Stable for a year or longer.

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. Rinse with 70% ethanol.
  3. Place in solution A for 1 hour.
  4. Wash off excess stain with 70% ethanol.
  5. Rinse well with tap water.
  6. Counterstain with nuclear fast red-tartrazine.
  7. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Mast cell granules  –  deep blue
  • Nuclei  –  red
  • Background  –  yellow

Notes

  • The staining solution is a modification of Gomori’s aldehyde fuchsin using toluidine blue instead of basic fuchsin.
  • Staining time may need to be increased as the solution ages (up to 2 hours). If staining takes longer than 2 hours, prepare a new solution.
  • Elastic fibres are unstained, likely because basic fuchsin can form dipole-dipole interactions and toluidine blue generally does not.
  • Mucins are stained very pale blue.
  • Different samples of this dye may vary in effectiveness. If a sample gives pale staining, try one from another vendor. Toluidine blue from Fisher Scientific was used to develop the method.

Safety Note

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

References

  1. Llewellyn, B. D., unpublished.
March 30, 2023
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Allen’s Stain for Mast Cells

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

Allen's Stain

for Mast Cells

11
steps
3
materials
print

Materials

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. Rinse with water.
  3. Stain nuclei lightly with alum hematoxylin.
  4. Rinse with tap water and blue hematoxylin.
  5. Rinse well with water.
  6. Place in neutral red for 10 minutes.
  7. Rinse with distilled water.
  8. Differentiate with 70% ethanol up to 10 minutes.
  9. Dehydrate with 96% ethanol up to 5 minutes.
  10. Dehydrate with N-butanol up to 10 minutes.
  11. Clear with xylene and mount using a resinous medium.

Expected Results

  • Nuclei – blue
  • Mast cell granules = 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, (1976).
    Lynch’s Medical Laboratory Technology., Ed. 3. Vol. II. pp. 980
    W. B. Saunders Company, Toronto, Canada.
  2. Putt, F A, (1972).
    Manual of Histopathological Staining Methods., pp. 233
    John Wiley & Sons, London, UK.
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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