Category

Intracytoplasmic Granules

Toluidine Blue for Nissl bodies

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

Toluidine Blue

for Nissl bodies

7
steps
7
materials
print

Materials

  • Staining Solution
    • Option 1
      MaterialAmount
      Toluidine blue1g
      Sodium tetraborate1g
      Distilled water100mL
    • Option 2
      MaterialAmount
      Thionin0.1g
      Distilled water100mL
  • Gothard’s Differentiator
    MaterialAmount
    Creosote50mL
    Cajeput oil40mL
    Xylene50mL
    Ethanol, absolute160mL

Tissue Sample

10µ 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 the staining solution at 56°C for at least 30 minutes
    or up to overnight at room temperature.
  3. Rinse well with running tap water.
  4. Rinse with absolute ethanol.
  5. Differentiate with Gothard’s differentiator, controlling microscopically.
  6. Rinse well with absolute ethanol.
  7. Clear with xylene and mount using a resinous medium.

Expected Results

  • Nissl bodies  –  blue
  • Nuclei  –  blue
  • Background  –  pale to unstained

Notes

  • Disbrey omits the sodium tetraborate from the toluidine blue solution and stains at room temperature, but recommends overnight staining when possible.
  • Methylene blue may be substituted for thionin.
  • Culling recommends diluting the Gothard’s differentiator with an equal volume of absolute ethanol before use.

Safety Note

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

References

  1. Disbrey, B. D., (1970)
    Histological laboratory methods, p. 232.
    E. & S. Livingstone, Edinburgh and London, UK.
  2. Culling, C F A, Allison, R T, Barr, W T, (1985).
    Cellular pathology technique., Ed. 4.
    Butterworths, London, England.
March 30, 2023
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Nissl’s Methylene Blue for Nissl bodies

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

Nissl's Methylene Blue

for Nissl bodies

7
steps
5
materials
print

Materials

  • Cajeput oil
  • Staining solution
    MaterialAmount
    Castile soap1.75g
    Methylene blue3.75g
    Distilled water1L

    Dissolve the soap in the water. Add and dissolve the dye. Allow to ripen at least three months.

  • Differentiator
    MaterialAmount
    Aniline10mL
    Ethanol, 95%90mL

Tissue Sample

Ethanol fixation is preferred. Formalin fixed tissue may be suitable. Sections should be thicker than usual, and were originally to be free floating sections, likely free hand sections prepared without freezing.

Protocol

  1. Place sections in the staining solution in a watch glass.
  2. Heat gently until bubbles appear.
  3. Transfer to the differentiating fluid in another watch glass.
  4. Differentiate until color ceases to be extracted.
  5. Transfer the section to a slide and gently blot dry.
  6. Clear with cajeput oil.
  7. Mount with a resinous medium.

Expected Results

  • Nissl  –  blue
  • Nuclei  –  blue

Notes

  • Nissl specified “Venetian soap”, which is the same as “Castile soap”. It refers to solid, bar soap made from olive oil and sodium hydroxide.

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. pp. 446.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
  2. Gatenby, J.B. and Beams, H.W., (1950)
    The Microtomist’s Vade-Mecum. 11 ed., pp. 508, para. 1099
    Churchill, London, UK.
March 30, 2023
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Kurnick Stain for Plasma Cells

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

Kurnick Stain

for Plasma Cells

5
steps
3
materials
print

Materials

Stock pyronin

MaterialAmount
Pyronin Y2g
Distilled water100mL

Stock methyl green

MaterialAmount
Methyl green2g
Distilled water100mL

Working solution

MaterialAmount
Stock pyronin12.5mL
Stock methyl green7.5mL
Distilled water30mL

Storing stock solutions

The pyronin stock solution should be extracted with chloroform ten times to ensure purity. The methyl green stock solution should be extracted a minimum of six times, until the chloroform is no longer colored with any crystal violet. Store both solutions over a layer of chloroform.

Tissue Sample

Most fixatives should be satisfactory if fixation time is not extended, but avoid potassium dichromate or picric acid. Overnight with a 10% formalin variant should be satisfactory.

Protocol

  1. Bring sections to distilled water via xylene and ethanol.
  2. Place into the staining solution for 6 minutes in a coplin jar.
  3. Blot gently.
  4. Dehydrate with n-butanol, two changes of 5 minutes each.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei – green to blue-green
  • Plasma cell cytoplasm, Nissl substance, nucleoli – red
  • Other cell cytoplasm – pink to unstained

Notes

  • As with other methyl green-pyronin methods used for demonstrating nucleic acids, in critical applications a control section should be treated with a 0.1% solution of ribonuclease for one hour at 37°C to remove RNA, and another treated with distilled water alone. Both control sections should be compared with an untreated but stained section.

Safety Note

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

References

  1. Disbrey, B. D., (1970)
    Histological laboratory methods. pp.187-188.
    E. & S. Livingstone, Edinburgh and London, UK.
March 30, 2023
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Lendrum’s Phloxine Tartrazine for Viral Inclusion Bodies

By Intracytoplasmic Granules, Paneth Cells, Protocols, Stain Target, Stain Type, Trichrome Staining, Yellowsolve Staining
Protocol

Lendrum's Phloxine Tartrazine

for Viral Inclusion Bodies

11
steps
6
materials
print

This method is also used for the demonstration of Paneth cell granules, and may be used as a substitute for the HPS if the differentiation in the tartrazine solution is shortened to retain pink cytoplasm and muscle.

Materials

  • Mayer’s hemalum
  • Solution A
    MaterialAmount
    Phloxine B0.5g
    Calcium chloride0.5g
    Distilled water100mL
  • Solution B
    MaterialAmount
    Tartrazineto saturation
    2-Ethoxy ethanol (cellosolve)100mL

Tissue Sample

5µ paraffin sections of formal sublimate fixed tissue is preferred. Formalin fixed tissue is suitable. Other fixatives are likely to be satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei to medium density with hemalum.
  3. Wash in tap water for 5 minutes. Blueing takes place in the phloxine solution.
  4. Place in solution A for 20 minutes.
  5. Rinse in tap water, blot almost dry. Some technologists rinse with cellosolve instead of blotting. The object is to remove all traces of water, as it interferes with the ability of tartrazine to extract phloxine and counterstain.
  6. Rinse with solution B to remove remaining water. Discard solution.
  7. Place in solution B until inclusions are red and all other tissue is yellow. The time varies considerably. Control microscopically.
  8. Rinse thoroughly but briefly with absolute ethanol.
    Do not rinse with water at this stage as it rapidly removes the tartrazine. Some technologists use cellosolve instead of ethanol.
  9. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Acidophil virus inclusion bodies  –  red
  • Paneth cell granules  –  red
  • Background  –  yellow

Notes

  • The 2-ethoxy ethanol must not be replaced by any other solvent.
  • The 2-ethoxy ethanol must be, and must remain, completely anhydrous.

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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Hitchcock Ehrich for Plasma Cells

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

Hitchcock Ehrich

for Plasma Cells

5

steps
3

materials
print

This method uses malachite green and acridine red to stain plasma cells in a similar manner to the methyl green pyronin methods.


Materials

Solution A

MaterialAmount
Malachite green1g
Distilled water100mL

Solution B

MaterialAmount
Acridine red3g
Distilled water100mL

Combine 1 part of solution A with 3 parts of solution B immediately before use.

Tissue Sample

Zenker fixation is recommended. Other fixatives may not be satisfactory.


Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into the staining solution for 30 seconds.
  3. Rinse with water.
  4. Dehydrate rapidly with absolute ethanol.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  green
  • Plasma cell cytoplasm  –  crimson
  • Other cell cytoplasm  –  pink

Safety Note

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


References

  1. Biological Staining Methods, 6th ed. (1957)
    Gurr George T.,
    George T. Gurr, London, UK
March 30, 2023
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Romanowsky – Giemsa General Oversight Stain

By Eosinophils, Intracytoplasmic Granules, Protocols, Stain Target
Protocol

Romanowsky – Giemsa

General Oversight Stain

9
steps
3
materials
print

Materials

  • Stock Giemsa or other Romanowsky stain e.g. Lieshman, Wright etc.
  • 0.1% acetic acid in distilled water.

Working Solution

MaterialAmount
Stock Romanowsky stain1mL
Distilled water or pH 6.8 buffer9mL

Make the diluted solution just before using. Discard after a few hours.

Tissue Sample

Most fixatives permit staining but the results may vary. 3 µ paraffin sections of neutral buffered formalin fixed tissue are usually suitable.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Optionally, treat with pH 6.8 phosphate buffer for 30 minutes.
  3. Place into the staining solution for 1 hour.
  4. Rinse well with water.
  5. Differentiate with acetic acid until nuclear morphology is clear. Control microscopically.
  6. Rinse well with distilled water.
  7. Blot dry with filter paper, then flood with xylene.
  8. Repeat step 6 until section is transparent (usually 4-5 times).
  9. Mount with a synthetic resinous medium.

Expected Results

  • Nuclei  –  blue
  • Cytoplasm  –  pink

Notes

  • Giemsa is usually diluted 1 in 10. Lieshman, Wright and others are often diluted 1 in 3.
  • Pretreatment with a pH 6.8 phosphate buffer is sometimes recommended immediately before placing in diluted stain. In that case, buffer with the same pH should be used to dilute the stock Romanowsky stain.
  • Differentiation may also be carried out in the same buffer as used to dilute the stock stain, but may take some time.
  • Drury and Wallington use pH 5.0 buffer mixed equal parts with methanol, and specify green Euparal as the mounting medium.
  • Sections may be rapidly dehydrated with ethanol instead of blotting, but this removes some blue staining and will likely destroy any metachromasia.
  • Sections stained with Romanowsky stains do not usually show the same range of colors that are shown by the same stain on smears, and the choice of stock Romanowsky stain does not necessarily influence the final appearance.

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., Alison, R.T. and Barr, W.T. (1985)
    Cellular Pathology Technique, 4th ed.
    Butterworths, London, UK.
  2. 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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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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Schmorl’s Stain for Reducing Substances

By Intracytoplasmic Granules, Melanin & Enterochromaffin, Metal Impregnation, Metal Impregnation, Non-Silver, Protocols, Stain Target, Stain Type
Protocol

Schmorl's Stain

for Reducing Substances

6
steps
4
materials
print

Materials

  • Nuclear fast red
  • Schmorl’s solution
    MaterialAmount
    Ferric chloride, 1% aqueous, fresh30mL
    Potassium ferricyanide, 1% aqueous, fresh4mL
    Distilled water6mL

This solution should be freshly prepared immediately 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 distilled water via xylene and ethanol.
  2. Place into freshly made Schmorl’s solution for 10 minutes.
  3. Wash well with water.
  4. Counterstain with nuclear fast red.
  5. Rinse well with water.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Reducing substances  –  blue
  • Nuclei  –  red

Notes

  • Reducing substances which may be present are colored blue. This includes melanin, for which it is a useful method, enterochromaffin and lipofuscin.
  • The Schmorl’s solution is from Lillie’s modification of Schmorl’s ferricyanide reduction method for tissue reducing substances.

Safety Note

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

References

  1. Lillie, R.D., (1954)
    Histopathologic technique and practical histochemistry Ed.2
    Blakiston, New York, USA.
March 30, 2023
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Trevan and Sharrock’s Methyl Green – Pyronin

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

Trevan and Sharrock's

Methyl Green – Pyronin

6
steps
8
materials
print

Materials

Methyl green stock

MaterialAmount
Methyl green2g
Distilled water100mL

Purifying the methyl green

Before making the methyl green-pyronin stock solution the methyl green must be purified to remove any methyl violet which has formed.

  1. Place the 2% aqueous stock methyl green solution in a stoppered separating funnel.
  2. Add some chloroform and shake periodically for about a half hour.
  3. Crystal violet will discolor the chloroform as it is removed.
  4. Allow to separate, then drain off the methyl green and discard the chloroform.
  5. Repeat the extraction until the chloroform is colorless or almost so.

Pyronin Y stock

MaterialAmount
Pyronin Y5g
Distilled water100mL

Acetate buffer pH4.8

MaterialAmount
0.1M sodium acetate119mL
0.1M acetic acid81mL

Methyl green–pyronin stock

MaterialAmount
Methyl green, 2% aqueous, purified10mL
Pyronin Y, 5% aqueous17.5mL
Distilled water250mL

Staining solution

MaterialAmount
Methyl green–pyronin stock1volume
Acetate buffer pH 4.81volume

Make fresh.

Tissue Sample

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

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Rinse with distilled water, then gently blot dry.
  3. Place into the staining solution for 20–30 minutes,
  4. Rinse with distilled water, then gently blot dry.
  5. Dehydrate rapidly with acetone.
  6. Clear with xylene and mount with a resinous medium.

Expected Results

  • DNA  –  blue–green
  • RNA  –  Red

Notes

  • Mercuric chloride fixation may cause the DNA to depolymerize during fixation and should be avoided. The differential staining depends on differences in the degree of polymerization of the two nucleic acids and depolymerization may cause the DNA to stain with pyronin.
  • Acid decalcified tissues are often unsatisfactory and may stain excessively red with pyronin. EDTA decalcification is usually satisfactory.
  • The extracted solutions of methyl green are reasonably stable, but changes in the expected nuclear coloration may require a freshly extracted solution of methyl green to be prepared.
  • This method is reasonably selective for nucleic acids but should be controlled with ribonuclease and/or deoxyribonuclease enzyme extractions if specificity is required.
  • Since this technique is quite simple, it is often recommended for plasma cells. Nissl substance also stains well.

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. 4, p. 164.
    Oxford University Press, London, England.
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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