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Nassar & Shanklin Impregnation for Reticulin

By Metal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type
Protocol

Nassar & Shanklin Impregnation

for Reticulin

22
steps
12
materials
print

Materials

  • Silver nitrate, 10% aqu.
  • Strong ammonium hydroxide (s.g. 0.880).
  • Oxalic acid, 2% aqu.
  • Yellow gold chloride, 2% aqu.
  • Sodium thiosulphate, 5% aqu.
  • Progressive hemalum
  • Mallory bleach
    MaterialAmount
    Potassium permanganate, 1% aqu.1vol
    Sulphuric acid, 0.5% aqu.1vol
  • Pyridinised silver nitrate
    MaterialAmount
    Silver nitrate, 2% aqu.10mL
    Pyridine3 drops
  • Reducer
    MaterialAmount
    Formalin, 2% aqu. neutralised1vol
    Ethanol, 95%1vol

Preparation of Ammoniacal Silver

  1. Place 1 mL of strong ammonium hydroxide into a flask.
  2. Pour in 7 mL of 10% aqueous silver nitrate.
  3. Add drops of 10% silver nitrate until a persistent, pale opalescence remains after shaking.
  4. Add an equal volume of distilled water. Add 3 drops pyridine for each 10 mL of solution for use.

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Oxidise with the Mallory bleach for 1-2 minutes.
  3. Rinse with distilled water.
  4. Bleach in Oxalic acid for 1-2 minutes.
  5. Rinse with distilled water.
  6. Rinse with tap water for 5 minutes.
  7. Rinse with 95% ethanol.
  8. Place in 2% silver nitrate
  9. Rinse with 95% ethanol.
  10. Treat with pyridinised silver nitrate at 50°C for 30 to 60 minutes.
  11. Rinse briefly with 95% ethanol.
  12. Treat with ammoniacal silver at 50°C for 5 minutes.
  13. Rinse briefly with 95% ethanol.
  14. Treat with the reducer for 2 minutes.
  15. Rinse well with distilled water.
  16. Tone with gold chloride solution until sections are grey.
  17. Rinse well with distilled water.
  18. Fix in 5% sodium thiosulphate for 2 minutes.
  19. Wash well with running tap water.
  20. Counterstain with a progressive hemalum and blue.
  21. Wash with tap water.
  22. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Nuclei  –  blue
  • Background  –  grey

Notes

  • Ensure that the ammonium hydroxide is fresh and full strength. Keep well stoppered when not in use. Remove sufficient for use from the stock bottle then immediately restopper.
  • 2% formalin is made by diluting strong formalin 1:50 with tap water (2 mL strong formalin, 98 mL tap water). The strong formalin used to make this should be neutralised, but do not use buffered formalin. Neutral formalin in this context may be made by keeping strong formalin over marble chips. However, be very careful as the gas given off may increase the pressure inside the container and cause an explosion. Either apply a cap loosely so gas can escape, or use a fermentation lock.
  • Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black reticulin fibres on a pale grey-brown background. Others tone longer (a few minutes) to produce black reticulin fibres on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.

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.
    Oxford University Press, London, England.
March 30, 2023
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Neutral Red for Nuclei

By Dye Type, Hematoxylin Alternatives, Protocols, Stain Type

Protocol

Neutral Red

for Nuclei

4
steps
3
materials
print

Materials

Staining Solution

MaterialAmount
Neutral red1g
Acetic acid, glacial1drop
Distilled water100mL

To avoid clumping, dust the dye onto the surface of the water while shaking it. Leave overnight, add the acid, and filter.

Tissue Sample

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

Protocol

  1. Complete the primary stain.
  2. Place into the solution for 1 – 3 minutes.
  3. Rinse well with water.
  4. Dehydrate with ethanol and clear with xylene.

Expected Results

  • Nuclei  –  red
  • Other tissues  –  as the primary stain

Notes

  • The small amount of acetic acid slightly acidifies the solution and reduces background staining, making the solution more selectively nuclear. Do not add more than specified.
  • The ethanol used for dehydration removes some of the stain. This permits a small degree of control.
  • If necessary, staining can be removed with acid alcohol. However, check that the primary stain will not be affected before doing so.

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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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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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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Laidlaw’s Impregnation for Reticulin on Bouin Fixed Tissue

By Metal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type
Protocol

Laidlaw's Impregnation

for Reticulin on Bouin Fixed Tissue

20
steps
10
materials
print

Materials

  • Iodine, 1% in 95% ethanol
  • Sodium thiosulphate, 5% aqu.
  • Potassium permanganate, 0.5% aqu.
  • Oxalic acid, 5% aqu.
  • Formalin, 1% aqu.
  • Yellow gold chloride, 0.2% aqu.
  • Silver nitrate, 60% aqu.
  • Strong ammonium hydroxide (s.g. 0.88)
  • Lithium carbonate, saturated aqu.
  • Neutral red, 1% aqu.

Preparation of Ammoniacal Silver

  1. Place 16 mL of the 60% silver nitrate in a flask.
  2. Add 185 mL of saturated lithium carbonate.
  3. Allow the precipitate to settle and decant the supernatent.
  4. Wash the precipitate with water, allow to settle, and decant several times.
  5. Add 60 mL distilled water.
  6. Add drops of strong ammonium hydroxide until the precipitate just redissolves.
  7. Dilute to 100 mL with distilled water.

Tissue Sample

5 µ paraffin sections of Bouin’s fluid fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended. The method was intended for staining reticulin in skin, but there is no reason it would not be satisfactory for other tissues.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place in alcoholic iodine for 3 minutes.
  3. Rinse well with tap water.
  4. Bleach with sodium thiosulphate for 3 minutes.
  5. Oxidise with potassium permanganate for 3 minutes.
  6. Rinse well with tap water.
  7. Bleach in Oxalic acid for a 5 minutes.
  8. Rinse well with tap water.
  9. Rinse with distilled water.
  10. Treat with ammoniacal silver for 10 minutess.
  11. Rinse with distilled water.
  12. Reduce in formalin for 10 minute.
  13. Rinse well with tap water.
  14. Rinse with distilled water.
  15. Tone with 0.2% gold chloride for 10 minutes.
  16. Rinse with distilled water.
  17. Wash well with running tap water.
  18. Counterstain with neutral red for 1 minute.
  19. Rinse with tap water.
  20. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Nuclei  –  red
  • Background  –  grey

Notes

  • Note that this method does not say to fix the impregnation with sodium thiosulphate. If sections fade or fixing the impregnation is considered desirable, then Place in 5% sodium thiosulphate for 5 minutes after step 16.
  • Ensure that both the ammonium hydroxide and sodium hydroxide are fresh and full strength. Keep both well stoppered when not in use. For the ammonium hydroxide, pour sufficient for use from the stock bottle into a beaker, then immediately restopper the stock bottle. Do not return excess ammonium hydroxide to the stock bottle.
  • After making the ammoniacal silver solution, smell the solution to ensure it has only a faint smell of ammonia. If the smell of ammonia is strong it indicates that too much ammonium hydroxide has been added. If so, it is preferable to make the solution again. Improperly made ammoniacal silver solutions can affect the quality of the impregnation.
  • 1% formalin is made by diluting strong formalin 1:100 with tap water (1 mL strong formalin, 99 mL tap water).
  • Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black reticulin fibres on a pale grey-brown background. Others tone longer (a few minutes) to produce black reticulin fibres on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.

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.
March 30, 2023
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La Manna’s Iron Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

La Manna's Iron Hematoxylin

8
steps
3
materials
print

Materials

MaterialAmountFunction
Hematoxylin1 gDye
Ferric ammonium sulfate3 gMordant
Distilled water100 mLSolvent

Compounding Procedure

  1. Dissolve the iron alum in half the water.
  2. Dissolve the hematoxylin in the rest.
  3. Combine.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse with tap water.
  4. Dip briefly in 1% hydrochloric acid in 70% ethanol.
  5. Wash well in running tap water to blue.
  6. Rinse with distilled water.
  7. Counterstain if desired.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Background  –  as counterstain or unstained

Notes

  • The simplicity of the formula suggests the solution is not stable for long.
  • The staining time should be determined by trial.

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:
    La Manna, (1937)
    Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik,
    v. 22, p. 55. Leipzig.
March 30, 2023
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Langeron’s Alum Hematoxylin Variants

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Langeron's Alum Hematoxylin Variants

8
steps
9
materials
print

Both of Langeron’s formulae are modifications of Mayer’s formulae. Langeron’s 1924 formula is a modification of Mayer’s 1896 formula, and Langeron’s 1942 formula is a modification of Mayer’s 1901 formula.

Langeron’s 1942 formula is the solution usually meant when Mayer’s hemalum is specified.

Materials

MaterialSolutionFunction
19241942
Hematoxylin4 g1 gDye
Ammonium alum50 g–Mordant
Potassium alum–50 gMordant
Distilled water700 mL1 LSolvent
Glycerol300 mL–Stabilizer
Sodium iodate–0.2 gOxidant
Glacial acetic acid20 mL–Acidifier
Citric acid–1 gAcidifier
Chloral hydrate–50 gAcidifier

Compounding Procedures

1924

  1. Grind the hematoxylin with glycerol in a pestle and mortar.
  2. Dissolve the other ingredients in water.
  3. Add the hematoxylin paste.
  4. Wash out the hematoxylin paste with the solution.
  5. Leave months to ripen.

1942

  1. Dissolve the Alum and dye in the water.
  2. Add the other ingredients.
  3. Bring to the boil.
  4. Cool to room temperature.
  5. The solution may be used immediately.
  6. Ammonium Alum may be substituted for potassium alum.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse well with water.
  4. Differentiate the 1924 solution with acid ethanol if necessary.
  5. Rinse with water and blue.
  6. Rinse well with water.
  7. Counterstain if desired.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • The 1924 solution is regressive, and staining time should be determined by trial.
  • The 1942 solution is progressive. Staining time is 5-10 minutes.
  • Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
  • Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

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.
March 30, 2023
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Launoy’s Alum Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Launoy's Alum Hematoxylin

8
steps
3
materials
print

Materials

MaterialAmountFunction
Hematein10 gDye
Potassium alum5 gMordant
Distilled water1 LSolvent

Compounding Procedure

  1. Combine all ingredients, and dissolve.
  2. The solution may be used immediately.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse well with water.
  4. Differentiate with acid ethanol if necessary.
  5. Rinse with water and blue.
  6. Rinse well with water.
  7. Counterstain if desired.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • This solution uses hematein instead of hematoxylin.
  • Although the staining characteristics are not given, the high dye and low Alum content would indicate it is likely a strong, regressive solution.
  • The staining time should be determined by trial.
  • Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
  • Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

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.
March 30, 2023
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Lee’s Alum Hematoxylin

By Dye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type
Protocol

Lee's Alum Hematoxylin

6
steps
5
materials
print

Materials

MaterialAmountFunction
Hematoxylin1 gDye
Ammonium alum50 gMordant
Distilled water1 LSolvent
Chloral hydrate50 gStabiliser
Sodium iodate0.2 gOxidant

Compounding Procedure

  1. Combine all the ingredients.
  2. Bring to a boil.
  3. Cool and filter.
  4. The solution may be used immediately.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 5-10 minutes.
  3. Rinse with water and blue.
  4. Rinse well with water.
  5. Counterstain if desired.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • The solution is similar to Mayer’s hemalum and should have much of the same characteristics.
  • Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

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.
March 30, 2023
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Lendrum & Mcfarlane Iron Alum-Celestine Blue for Nuclei

By Dye Type, Hematoxylin Alternatives, Protocols, Stain Type
Protocol

Lendrum & Mcfarlane Iron Alum-Celestine Blue

for Nuclei

6
steps
5
materials
print

Materials

  • An eosin solution, or other counterstain
  • Iron alum-celestine blue
    MaterialAmount
    Ferric ammonium sulfate5g
    Celestine blue B0.5g
    Distilled water100mL
    Glycerol14mL

Preparation

  1. Dissolve the ferric ammonium sulfate in the distilled water.
  2. Add the celestine blue B.
  3. Boil for 5 minutes.
  4. Cool and filter.
  5. Add the glycerol.

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 the iron alum-celestine blue solution for 3 minutes to 2 hours.
  3. Wash with tap water.
  4. Counterstain with eosin or other counterstain.
  5. Dehydrate with 95% and absolute ethanols.
  6. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Cytoplasm  –  pink

Notes

  • The iron alum-celestine blue solution is stable for a few months.
  • A staining time of 5-10 minutes is usually satisfactory for formalin fixed tissues.
  • This method has been recommended as a substitute for Hematoxylin and Eosin.
  • The celestine blue solution is a modification of that recommended by Proescher and Arkush, and contains added glycerol.
  • Gallamine blue and gallocyanin have also been recommended in this method. Neither dye is as stable as celestine blue, although the colour with gallocyanin most closely resembles alum hematoxylin of all three dyes.

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:
    Lendrum and McFarlane, (1940)
    Journal of Pathology and Bacteriology, v. 50, pp. 381
March 30, 2023
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