Skip to main content
search
Category

Nissl Bodies

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
Love0

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
Love0

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
Love0

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
Love0

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
Love0