Skip to main content
search
Category

Protocols

[facetwp template="protocol_by_facet"]

Maresch’ Impregnation for Reticulin in Liver

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

Maresch' Impregnation

for Reticulin in Liver

12
steps
8
materials
print

Materials

  • Silver nitrate, 2% aqu.
  • Silver nitrate, 10% aqu.
  • Sodium hydroxide, 40% aqu.
  • Strong ammonium hydroxide (s.g. 0.88).
  • Formalin, 20% aqu.
  • Yellow gold chloride, 0.2% in 0.2% acetic acid.
  • Sodium thiosulphate, 5% aqu.
  • Neutral red, 1% aqu.

Preparation of Maresch’ Ammoniacal Silver

  1. Place 100 mL of 10% silver nitrate in a flask.
  2. Add 0.5 mL of 40% sodium hydroxide and mix well.
  3. Add drops of strong ammonium hydroxide until the precipitate just dissolves.

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. Place in 2% silver nitrate for 12-24 hours.
  3. Treat with ammoniacal silver solution for 2-30 minutes.
  4. Rinse well with distilled water.
  5. Place in 10% formalin for 1 hour.
  6. Wash well with tap water.
  7. Rinse with distilled water.
  8. Tone with acidified gold chloride solution for 1 hour.
  9. Rinse well with distilled water.
  10. Fix in 5% sodium thiosulphate for 10-15 minutes.
  11. Wash well with tap water.
  12. Dehydrate with ethanol, clear with carbol-xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Background  –  grey

Notes

  • Ensure that the ammonium hydroxide is fresh and full strength. Keep well stoppered when not in use. After removing the amount required immediately restopper the bottle.
  • Improperly made ammoniacal silver solutions can affect the quality of the impregnation. There should be a faint, persistent opalescence, with only a faint smell of ammonia.
  • 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
Love0

Martinotti’s Alum Hematoxylin

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

Martinotti's Alum Hematoxylin

8
steps
5
materials
print

Materials

MaterialAmountFunction
Hematein2 gDye
Ammonium alum15 gMordant
Distilled water700 mLSolvent
Methanol150 mLSolvent
Glycerol150 mLStabiliser

Compounding Procedure

  1. Dissolve the Alum in 600 mL water.
  2. Dissolve the hematein in 100 mL water.
  3. Combine, and add the other ingredients.
  4. 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.
  • 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
Love0

Masson’s Alum Hematoxylin

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

Masson's Alum Hematoxylin

8
steps
4
materials
print

Materials

MaterialAmountFunction
Hematein20 gDye
Potassium alum60 gMordant
Distilled water1 LSolvent
Glacial acetic acid20 mLAcidifier

Compounding Procedure

  1. Dissolve the Alum in boiling water.
  2. Add the dye.
  3. Filter when cool.
  4. Add acetic acid.
  5. 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
  • The high concentration of hematein indicates this is most 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
Love0

Masson’s Iron Hematoxylin

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

Masson's Iron Hematoxylin

8
steps
3
materials
print

Materials

Solution A

MaterialAmountFunction
Ferric ammonium sulfate4 gMordant
Distilled water100 mLSolvent

Solution B

MaterialAmountFunction
Hematoxylin1 gDye
Distilled water100 mLSolvent

Compounding Procedure

  1. Make each solution separately.
  2. Solution B should be ripened for a minimum of one month.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into solution A for 5-10 minutes preheated to 50°C.
  3. Rinse with distilled water.
  4. Place into solution B for 5-10 minutes preheated to 50°C.
  5. Rinse with tap water.
  6. Differentiate in solution A at room temperature, controlling microscopically.
  7. Wash well in running tap water to blue.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei and other structures  –  blue to black

Notes

  • This is a modification of Heidenhain’s iron hematoxylin, and differs solely in the concentration of the reagents, and the temperature at which staining is carried out.
  • The stock solutions are stable for some time.
  • The hematoxylin solution needs to be ripened.
  • The degree of differentiation will determine which tissue components are prominent. The method can demonstrate many structures, including chromosomes, nuclear components, mitochondria and muscle striations
  • The solutions may be reused, with the exception of the solution A used to differentiate, which should be fresh each time.
  • Counterstaining is not recommended.
  • This method is usually recommended for monochrome photography.

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:
    Heidenhain, M., (1892).
    Festschrift Herrn A. von Kolloker zur Feier seines fünfzigjährigen medicinischen
    Doktorjubiläums, p.118. Wilhelm Engellmans, Leipzig, Germany
    and:
    Masson, M.,, (1892).
    Bulletin et mémoires de la Société anatomique de Paris, v.87. p.291.
March 30, 2023
Love0

Maxwell’s Trichrome for Pituitary Cells

By Protocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step
Protocol

Maxwell's Trichrome

for Pituitary Cells

12
steps
12
materials
print

Materials

Solution A

MaterialAmount
Acid fuchsin1g
Distilled water100mL

Solution B

MaterialAmount
Ammonia (0.880)0.02mL
Distilled water100mL

Solution C

MaterialAmount
Hydrochloric acid (Conc.)0.1mL
Distilled water100mL

Solution D

MaterialAmount
Phosphomolybdic acid0.5g
Distilled water100mL

Solution E

MaterialAmount
Orange G2g
Aniline blue2g
Phosphomolybdic acid1g
Distilled water100mL

Maxwell’s fluid

MaterialAmount
Cedarwood oil30mL
Thyme oil40mL
Xylene15mL
Ethanol, absolute15mL

Tissue Sample

Paraffin sections at 5µ are suitable. Heidenhain’s (Zenker formol) fixation was specified. Other mercuric chloride fixatives would likely be satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into solution A for 30 minute.
  3. Rinse with distilled water.
  4. Place into solution B until differentiated.
  5. Place into solution C for a few seconds.
  6. Place into solution D for 3 minutes.
  7. Place into solution E for 60 minutes.
  8. Rinse with distilled water.
  9. Differentiate with 95% ethanol.
  10. Dehydrate with ethanol.
  11. Clear with Maxwell’s fluid or xylene.
  12. Mount with Canada balsam or other resinous medium.

Expected Results

  • Nuclei  –  red
  • Acidophils  –  orange red
  • basophils  –  blue

Notes

  • Maxwell’s fluid was originally specified for clearing, but this is probably not necessary.

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:
    Maxwell, (1938)
    Stain Technology, vol. 13, pp. 93
March 30, 2023
Love0

Sweat, Meloan & Puchtler’s One Step Trichrome

By Protocols, Stain Type, Trichrome Staining, Trichrome, One-Step
Protocol

Sweat, Meloan & Puchtler's

One Step Trichrome

7
steps
6
materials
print

Materials

Solution A

MaterialAmount
Chromotrope 2R0.6g
Aniline blue0.6g
Phosphomolybdic acid1g
Hydrochloric acid, conc.1mL
Distilled water100mL

Solution B

MaterialAmount
Acetic acid, glacial1mL
Distilled water100mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place in Bouin’s fixative at 56°C for one hour.
  3. Wash with running tap water 3 to 4 minutes to remove yellow.
  4. Place into solution A for 1 minute.
  5. Rinse with solution B for 30 seconds.
  6. Dehydrate rapidly with ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei, cytoplasm, muscle, fibrin & elastic  –  red
  • Collagen  –  blue

Notes

  • A nuclear stain is not used as the hydrochloric acid will remove it.

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.
March 30, 2023
Love0

Mitchell’s Alum Hematoxylin

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

Mitchell's Alum Hematoxylin

6
steps
4
materials
print

Mitchell’s formula is from 1883 and is now obsolete. It does, however, show how Alum hematoxylin solutions were originally prepared, and the variability inherent in the procedures for doing so. The modern formula should stain satisfactorily.

Materials

Original Formula

MaterialAmountFunction
Logwood, ground2 ouncesDye
Potassium alum9 ouncesMordant
Distilled wateras neededSolvent
Glycerol4 fl. ouncesStabiliser

Compounding Procedures

  1. Moisten the ground logwood with water and pack it into a funnel.
  2. Pour water onto the wood until it comes through barely coloured.
  3. Remove the wood from the funnel, spread out and dry.
  4. Dissolve the Alum in 8 fluid oz of water.
  5. Moisten the logwood with some Alum water and pack it tightly into the funnel.
  6. Pour the rest of the Alum solution onto the logwood.
  7. When the first drops come through, seal up the tip of the funnel.
  8. Leave 48 hours for the dye to be extracted.
  9. Remove the seal and collect the fluid that comes through.
  10. Pour on more water until 12 fluid oz have been collected.
  11. Add the glycerol, mix well and filter.

Modern Formula

MaterialAmountFunction
Hematoxylin3 gDye
Potassium alum35 gMordant
Distilled water340 mLSolvent
Glycerol115 mLStabiliser

Compounding Procedures

  1. Dissolve the Alum in the water.
  2. Add the hematoxylin and mix until dissolved.
  3. Add the glycerol, mix well and filter.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for a few minutes, or dilute 1:7 with distilled water and stain overnight.
  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 staining characteristics were not given, but it is likely progressive, especially when diluted. If overstaining does occur, differentiate with 0.5 – 1% hydrochloric acid in 70% ethanol.
  • The reason for the pre-wash of the logwood in water during the original preparation method was stated to be for the removal of tannin.
  • The amount of hematoxylin in the modern formula is based on the fact that logwood contains up to 10% of the dye. Two ounces (about 57g) could contain a maximum of about 6 grams of dye. A 50% extraction, or 3 grams hematoxylin has been allowed for. It could well have been more or less than this.
  • The original formula gives 255 grams of potassium Alum to dissolve in 227 mL water. It takes just over 7 mL water to dissolve 1 gram crystalline potassium alum, so the amount of water specified would be able to dissolve only about 32 grams.
  • 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. Arthur Bolles-Lee, (1885)
    The Microtomist’s Vade-Mecum
    Originally published by: J & A Churchill, London, England.
    Republished by: Science Heritage Ltd., Lincolnwood, Illinois, USA.
  2. Susan Budavari, Editor, (1996)
    The Merck Index, Ed. 12
    Merck & Co., Inc., Whitehouse Station, NJ, USA
  3. Lemmens, R. H. M. J. and Wulijarna-Soetjipto, N., Editors. (1992)
    Plant resources of South East Asia No. 3,
    Dye and tannin-producing plants.
    PROSEA, Bogor, Indonesia.
March 30, 2023
Love0

Molnar’s Alum Hematoxylin Variants

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

Molnar's Alum Hematoxylin Variants

8
steps
9
materials
print

Materials

MaterialVariation IVariation IFunction
Hematoxylin10 g4 gDye
Ammonium or potassium alum50 g50 gMordant
Distilled water1 L1 LSolvent
95% ethanol50 mLSolvent
Mercuric oxide5 gOxidant
Sodium iodate0.3 gOxidant
Glacial acetic acid20 mLAcidifier
Citric acid1.5 gAcidifier
Chloral hydrate75 gAntioxidant

Compounding Procedures

Variation I

  1. Dissolve the dye into the ethanol, and the Alum into the water.
  2. Combine the solutions in an Erlenmeyer flask and slowly add the mercuric oxide
  3. Reheat until the solution changes colour to a dark purple.
  4. Remove from heat and cool rapidly by swirling the flask in cold water.
  5. When cool, add the acetic acid.

Variation II

  1. Dissolve the Alum into the water.
  2. Add the hematoxylin, sodium iodate, citric acid and chloral hydrate in that order.
  3. Filter before use.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for the specified time.
    1. Var I for 10 minutes.
    2. Var II for 8-10 seconds.
  3. Rinse well with water.
  4. Differentiate Var I with acid ethanol for a few seconds.
  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

  • Var I was recommended for double embedded tissues.
  • The use of mercuric oxide as an oxidant is now deprecated due to its toxicity. Var I could be modified by using sodium iodate at about 0.3 grams instead.
  • 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. Molnar, L. N.,
    Modification of Harris hematoxylin for sections from tissue double embedded with nitrocellulose and paraffin.
    Histologic, v 5, Nº 1, January, 1975
  2. Molnar, L. N.,
    Modification of Mayer’s hematoxylin-eosin method.
    Histologic, v 6, Nº 4, October, 1976
March 30, 2023
Love0

Morel & Bassal’s Iron Hematoxylin

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

Morel & Bassal's Iron Hematoxylin

8
steps
6
materials
print

Materials

Solution A

MaterialAmountFunction
Hematoxylin1 gDye
95% ethanolmLSolvent

Solution B

MaterialAmountFunction
Ferric chloride2 gMordant
Cupric acetate0.04 gMordant
Distilled water100 mLSolvent
Hydrochloric acid1 mLAcidifier

Compounding Procedure

  1. Make each solution separately.
  2. For use, combine equal parts of solutions A and B.
  3. The working solution may be used immediately, but is not stable for long.

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 stock solutions are stable for some time.
  • The working solution should be made fresh, and is likely 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:
    Morel and Bassal, (1909)
    Journal de l’anatomie et de la physiologie normales et pathologique de l’homme
    et des animaux, v. 45, p. 632.
March 30, 2023
Love0

Murray’s Iron Hematoxylin

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

Murray's Iron Hematoxylin

8
steps
4
materials
print

Materials

Solution A

MaterialAmountFunction
Ferric ammonium sulfate3.5 gMordant
Distilled water100 mLSolvent

Solution B

MaterialAmountFunction
Hematoxylin0.5 gDye
Distilled water100 mLSolvent

Compounding Procedure

  1. Make each solution separately.
  2. Solution B should be ripened for a minimum of one month.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into solution A for 30 minutes to 24 hours.
  3. Rinse with distilled water.
  4. Place into solution B for 30 minutes to 24 hours.
  5. Rinse with tap water.
  6. Differentiate in solution A, controlling microscopically.
  7. Wash well in running tap water to blue.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei and other structures  –  blue to black

Notes

  • This is a modification of Heidenhain’s iron hematoxylin, and differs solely in the concentration of the reagents.
  • The stock solutions are stable for some time.
  • The hematoxylin solution needs to be ripened.
  • The degree of differentiation will determine which tissue components are prominent. This method can demonstrate many structures, including chromosomes, nuclear components, mitochondria and muscle striations
  • The solutions may be reused, with the exception of the solution A used to differentiate, which should be fresh each time.
  • Counterstaining is not recommended.
  • This method is usually recommended for monochrome photography.

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:
    Heidenhain, M., (1892).
    Festschrift Herrn A. von Kolloker zur Feier seines fünfzigjährigen medicinischen
    Doktorjubiläums, p.118. Wilhelm Engellmans, Leipzig, Germany
    and:
    Heidenhain, M.,, (1919).
    Annual report of the Cancer Research Fund, v.16. p.77. London, England.
March 30, 2023
Love0