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Intracytoplasmic Granules

Cresyl Violet Stain for Nissl Bodies

By Intracytoplasmic Granules, Nissl Bodies, Protocols, Stain Target
Protocol

Cresyl Violet Stain

for Nissl Bodies

11
steps
4
materials
print

Materials

Staining Solution

MaterialAmount
Cresyl violet1g
Distilled water100mL

Differentiator – Option 1

MaterialAmount
Ethanol, 95%100mL
Cajeput oila few drops

Differentiator – Option 2

MaterialAmount
Ethanol, 95%100mL
Acetic acid, glacial5 drops

Tissue Sample

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

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into the staining solution for 15-30 minutes.
  3. Rinse with water, dehydrate with absolute ethanol and clear with xylene.
  4. Leave in xylene for one hour.
  5. Rinse well with absolute ethanol.
  6. Bring one slide at a time to 95% ethanol.
  7. Differentiate in either cajeput ethanol or acetic ethanol, controlling microscopically.
  8. Rinse well with 95% ethanol to stop differentiation.
  9. Dehydrate with absolute ethanol.
  10. Clear with xylene
  11. Coverslip using a resinous medium.

Expected Results

  • Nissl bodies  –  blue-violet
  • Nuclei  –  blue violet

Notes

  • The first dehydration and clearing of the stained section (steps 3-4) improves the sharpness of diffentiation and is recommended.
  • Differentiation, as in steps 5-10, may be repeated if necessary.

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. 379.
    Oxford University Press, London, England.
  2. Bench Manual
March 30, 2023
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Einarson’s Gallocyanin-Chrome Alum

By Intracytoplasmic Granules, Nissl Bodies, Plasma Cells, Protocols, Stain Target
Protocol

Einarson’s

Gallocyanin-Chrome Alum

5
steps
3
materials
print

Materials

Solution

MaterialAmount
Chrome alum5g
Gallocyanin0.15g
Distilled water100mL

Solution Preparation

  1. Add the chrome alum and gallocyanin to the water.
  2. Bring to a boil and simmer for 20 minutes.
  3. Cool and filter.

Tissue Sample

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

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place in the staining solution for 24-48 hours.
  3. Rinse well with water.
  4. Dehydrate with ethanols.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nucleic acids  –  blue

Notes

  • Chrome alum is chromium potassium sulfate dodecahydrate, CrK(SO4)2·12H2O
  • Culling states that the pH is 1.64, and that changing this will eliminate or accentuate non-specific staining. Addition of up to 10 mL N hydrochloric acid will eliminate background staining, and addition of up to 5 mL N sodium hydroxide will accentuate it. In either case, staining of nucleic acids is not affected.

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:
    Einarson, (1932)
    American Journal of Pathology, v. 8, p. 295
    Boston, USA
  2. Culling C.F.A., (1974)
    Handbook of histopathological and histochemical techniques Ed. 3
    Butterworth, London, UK.
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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Lendrum’s Chromotrope 2R for Eosinophils

By Eosinophils, Intracytoplasmic Granules, Protocols, Stain Target
Protocol

Lendrum's Chromotrope 2R

for Eosinophils

6
steps
4
materials
print

Materials

Preparation

  1. Place the phenol in an Erlenmeyer flask and melt it under hot water through the glass.
  2. Add the chromotrope 2R and mix well into a sludge.
  3. Add the water and mix well.
  4. Filter before use.

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. Stain nuclei with Mayer’s hemalum and blue.
  3. Place in the staining solution for 30 minutes.
  4. Wash well with tap water.
  5. Dehydrate with ethanol.
  6. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Eosinophil granules  –  bright red
  • Erythrocytes  –  may be lightly stained
  • Paneth cell granules  –  may be brownish
  • Enterocromaffin granules  –  may be brownish

Notes

  • The basis of this method is difficult to rationalise. Phenol is acidic and thus lowers the pH. This is often used as the basis for explaining the method, but usually an acid dye stains all basic components of the tissue (muscle, cytoplasm, collagen) intensely when applied at an acid pH. With this method eosinophils are intensely stained, but other components that would usually stain with an acidified acid dye are not. Phenol can have an intensifying effect, as is clear from its inclusion in carbol fuchsin, when it intensifies staining with basic fuchsin, but the mechanism has not been satisfactorily explained.

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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Buffered Thionin for Nissl Bodies

By Intracytoplasmic Granules, Nissl Bodies, Protocols, Stain Target
Protocol

Buffered Thionin

for Nissl Bodies

16
steps
3
materials
print

Materials

MaterialAmount for pH 3.7 SolutionAmount for pH 4.5 Solution
Acetic acid, 0.6% (0.1M)90mL60mL
Sodium acetate, 0.8% (0.1M)10mL40mL
Thionin, 1% aqueous2.5mL2.5mL

Tissue Sample

10µ paraffin sections fixed in 10% formalin variants or Carnoy’s chloroform-ethanol-acetic mixture are suitable. Other fixatives may be satisfactory.


Protocol

Standard Method

  1. Bring sections to water via xylene and ethanol.
  2. Place into one of the staining solutions for 20-60 minutes.
  3. Dehydrate with ascending concentrations of ethanol.
  4. Clear with xylene and mount with a resinous medium.

Alternative Method

  1. Dilute the thionin with distilled water instead of acetate buffer.
  2. Bring sections to water via xylene and ethanols.
  3. Stain in aqueous thionin for 20-60 minutes.
  4. Rinse with ethanol, 50%.
  5. Differentiate with 0.25% acetic acid in 95% ethanol, controlling microscopically.
  6. Rinse well with 95% ethanol.
  7. Complete dehydration with absolute ethanol.
  8. Clear with xylene and coverslip using a resinous medium.

Expected Results

StructurepH 3.7 Staining SolutionpH 4.5 Staining Solution
Nissl bodiesbluedark blue
Nucleibluedark blue
Backgroundpale or unstainedpale blue

Safety Note

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


References

  1. Davenport, H.A.. (1960).
    Histological and Histochemical Technics,
    W. B. Saunders, Philadelphia, USA.
    Citing:
    Windle, W. F., Rhines, R. and Rankin, J. (1943),
    A Nissl method using buffered solutions of thionin.
     Stain Technology, v 8, pp. 77-86.
    and:
    Conn, H. J. and Darrow, M. A.,, (1946),
    Staining procedures.
    Biotech Publications, Geneva, New York.
March 30, 2023
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Roque’s Stain for Cell Inclusions

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

Roque's Stain

for Cell Inclusions

7
steps
6
materials
print

Materials

Stock solution A

MaterialAmount
Citrate buffer, pH 5.8100mL
Methyl green, purified0.1g
Thionin0.0165g

Dissolve the thionin in a small amount of water. Add the buffer and methyl green. Shake and filter. Use fresh.

Citrate buffer, pH 5.8

MaterialAmount
Hydrochloric acid, 0.01M42mL
Sodium citrate, 0.01M58mL

Dehydrant

MaterialAmount
Tertiary butanol80mL
Ethanol, absolute20mL

Tissue Sample

Fix 2mm thick pieces of tissue in 10% formalin containing 1% sodium acetate for 3 hours. Fix smears with methanol.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into the staining solution for 30 minutes at 40°C.
  3. Rinse briefly with distilled water.
  4. Place into dehydrant for 30 seconds
  5. Replace dehydrant, 2 changes, 3 minutes each.
  6. Rinse with absolute ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue-green
  • Nucleolar and cytoplasmic basophil substances  –  red-purple

Notes

  • The reference specifies methyl green, but gives the CI number for ethyl green.
  • The methyl green should be purified, but as a powder rather than as a solution:
    • Add about 10g methyl green to 200 mL chloroform in an Erlenmeyer flask.
    • Shake well.
    • Filter under vacuum and in a fume chamber.
    • Repeat until the chloroform is blue-green instead of violet.

Safety Note

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

References

  1. Humason, G. L., (1967).
    Animal Tissue Techniques., pp. 278
    W. H. Freeman and Company, San Francisco, CA, USA.
March 30, 2023
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Llewellyn’s Sirius Red for Amyloid

By Amyloid, Direct Dye Staining, Eosinophils, Intracytoplasmic Granules, Paneth Cells, Protocols, Stain Target, Stain Type
Protocol

Llewellyn's Sirius Red

for Amyloid

8
steps
3
materials
print

Materials

MaterialAmount
Sirius red F3B0.5g
Distilled water50mL
Ethanol, absolute50mL

Staining Solution Preparation

  1. Dissolve the dye into the water, add ethanol and mix well.
  2. Add 1 mL of 1% sodium hydroxide. Then, while strong backlighting and swirling, add drops of 20% sodium chloride until a fine haze is detected. Usually about 2 mL is adequate. Adding more than 4 mL causes excessive precipitation. The solution is reasonably stable for several months, but slowly deteriorates. Extend the staining time to compensate. When it requires more than 2 hours to adequately stain, prepare a new solution.

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. Stain nuclei with a progressive alum hematoxylin for a few minutes.
  3. Rinse with tap water.
  4. Rinse with ethanol.
  5. Place into alkaline sirius red for 1 – 2 hours.
  6. Rinse well with tap water.
  7. Dehydrate with absolute ethanol.
  8. Clear with xylene and mount with a resinous medium.

Expected Results

  • Amyloid  –  red
  • Eosinophil and Paneth cell granules  –  red
  • Nuclei  –  blue
  • Background  –  colorless
   

Notes

  • Amyloid displays deep green birefringence when viewed with crossed polarisers, one above and one below the section.
  • Eosinophils and Paneth cell granules are also demonstrated. If used for this purpose the sodium chloride may be ommitted.
  • This method uses sirius red F3B. The dye Sirius red 4B is not suitable.

Safety Note

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

References

  1. Llewellyn, B.D., (1970)
    An improved sirius red method for amyloid.
    Journal of Medical Laboratory Technology, v 23, 308
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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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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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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