Category

Stain Type

Roach & Smith’s Bismuth Hematoxylin

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

Roach & Smith's Bismuth Hematoxylin

4
steps
7
materials
print

This method demonstrates arginine.

Materials

MaterialAmountFunction
Hematoxylin50 mgDye
Ethanol, abs.5 mLSolvent
Sodium iodate10 mgOxidant
Bismuth nitrate50 mgMordant
Citric acid, 0.5M aqu.85 mLAcidifier
Glycerol20 mLSolvent
Sodium hydroxide, 1 M aqu.19 mLAlkaliser

Compounding procedure

  1. Dissolve the bismuth nitrate into the citric acid solution.
  2. Add 10 mL of the glycerol, then the sodium hydroxide solution.
  3. Adjust the pH to 5.2.
  4. Dissolve the hematoxylin into the ethanol.
  5. Combine the two solutions.
  6. Add the sodium iodate dissolved in 1 mL distilled water.
  7. Stir for 20 minutes.
  8. Add the remaining 10 mL of glycerol.

This solution has a usable life of about 3 hours.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into bismuth hematoxylin for 10 minutes.
  3. Rinse with water.
  4. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Arginine residues  –  black

Notes

  • The formula calls for bismuth nitrate pentahydrate, Bi(NO3)3.5H20.
  • This method demonstrates proteins with an arginine content of about 12% or more.
  • Nuclear histones and myelin basic protein stain strongly.
  • The authors suggest that the bismuth binds to the guanidine groups of arginine.

Safety Note

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

References

  1. Roach, J. B., and Allen, A. A., (1997)
    Bismuth hematoxylin stain for arginine residues.
    Biotechnic & Histochemistry, v.72, Nº 1, p. 49.
March 30, 2023
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Proescher & Arkush Iron Alum-Celestine Blue for Nuclei

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

Proescher & Arkush Iron Alum-Celestine Blue

for Nuclei

6
steps
4
materials
print

Materials

  • An eosin solution, or other counterstain
  • Iron alum-celestine blue
    MaterialAmount
    Ferric ammonium sulphate5g
    Celestine blue B0.5g
    Distilled water100mL

    Dissolve the ferric ammonium sulphate in the distilled water. Add the celestine blue B. Boil for 5 minutes. Cool and filter.

Tissue Sample

5 µ 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.
  • Proescher & Arkush also recommended the dyes gallamine blue and gallocyanin. Neither dye is as stable as celestine blue, although the color 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:
    Proescher and Arkush, (1928)
    Stain Technology, v. 3, pp. 36
March 30, 2023
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Gram Churukian–Schenk for Gram Positive & Negative Bacteria

By Bacteria, Gram Staining, Protocols, Stain Target, Stain Type
Protocol

Gram Churukian–Schenk

for Gram Positive & Negative Bacteria

14
steps
15
materials
print

Materials

  • Stock basic fuchsin
    MaterialAmount
    Basic fuchsin0.5g
    Distilled water100mL
  • Solution A
    Crystal violet 10% in 2mL ethanol

    MaterialAmount
    Ammonium oxalate 1% aqueous98mL
  • Solution B
    MaterialAmount
    Iodine2g
    Potassium iodide4g
    Distilled water400mL
  • Solution C
    MaterialAmount
    Absolute ethanol1volume
    Acetone1volume
  • Solution D
    MaterialAmount
    Stock basic fuchsin5mL
    Distilled water45mL
  • Solution E
    MaterialAmount
    Picric acid0.1g
    Acetone100mL
  • Solution F
    MaterialAmount
    Acetone1Volume
    Xylene1volume

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 in solution A for 2 minutes.
  3. Rinse with tap water.
  4. Place in solution B for 1 minute.
  5. Rinse well with tap water.
  6. Blot the slide, but not the tissue.
  7. Decolorise with solution C until no more blue floods off.
  8. Wet section with solution D then apply for 1 minute.
  9. Rinse with distilled water.
  10. Blot the slide, but not the tissue.
  11. Place in acetone for 3 seconds.
  12. Differentiate in solution E for 10 seconds.
  13. Quickly dip a few times in solution F.
  14. Clear with xylene and mount with a resinous medium.

Expected Results

  • Gram positive bacteria  –  blue
  • Nocardia and actinomyces  –  blue, or blue and red
  • Gram negative bacteria  –  red
  • Nuclei, Elastic, Paneth cells  –  red
  • Background  –  yellow

Notes

  • Picric acid should be handled with care. Solution E may be made by taking 12 mL of a saturated solution of picric acid in ethanol and diluting to 1 liter with acetone.
  • Basic fuchsin homologues with CI numbers of 42500 (pararosanilin) or 42510 (rosanilin) were specified. It was also noted that new fuchsin (CI 42520) was satisfactory, but not recommended because it was not certified by the Biological Stain Commission.
  • The authors note that sections must not be allowed to dry out after being stained with basic fuchsin. Doing so makes it difficult or impossible to properly differentiate the red counterstain.

Safety Note

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

References

  1. Churukian, C. J. & Schenk, E. A. (1982)
    A method for demonstrating Gram-positive and Gram-negative bacteria.
    Journal of Histotechnology, v.5, No.3, p.127
March 30, 2023
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Gram Weigert for Fibrin and Gram Positive Bacteria

By Bacteria, Fibrin, Gram Staining, Protocols, Stain Target, Stain Type
Protocol

Gram Weigert

for Fibrin and Gram Positive Bacteria

11
steps
9
materials
print

Materials

Eosin

MaterialAmount
Eosin Y ws5g
Distilled water100mL

Crystal violet

MaterialAmount
Crystal violet1g
Distilled water100mL

Gram’s iodine

MaterialAmount
Iodine2g
Potassium iodide4g
Distilled water400mL

Aniline-Xylene

MaterialAmount
Aniline1volume
Xylene1volume

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 in eosin solution for 5 minutes.
  3. Rinse with tap water.
  4. Place in crystal violet 1 minute.
  5. Rinse with tap water.
  6. Flood with Gram’s iodine for 1 minute.
  7. Rinse with tap water.
  8. Gently blot the section, being careful not to damage it.
  9. Decolorise the section with aniline-xylene.
  10. Rinse with several changes of xylene to remove all aniline.
  11. Mount with a resinous medium.

Expected Results

  • Gram positive bacteria  –  blue
  • Fibrin  –  blue
  • Background  –  pink

Notes

  • Control the differentiation with aniline-xylene microscopically. To examine, place the section in xylene to stop dye removal. Return to aniline-xylene if more differentiation is needed. Stop differentiation when the target element has good contrast.
  • Increasing the aniline content of the aniline-xylene will increase the speed of dye removal. Decreasing it will slow dye removal.
  • If the background is not pink enough, increase the time in eosin, stain in eosin at elevated temperature, or increase the eosin concentration.
  • The eosin counterstain may be omitted entirely if wished.

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., (1963)
    Handbook of histopathological techniques, 2nd ed.
    Butterworths, London.
  2. McManus, J.F.A. and Mowry, R.W., (1960),
    Staining methods, histologic and histochemical,
    Harper & Row, New York, NY, USA.
March 30, 2023
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Baker’s Hematal Variants

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

Baker's Hematal Variants

15
steps
11
materials
print

Materials

MaterialSolutionFunction
Stock AStock B
Aluminum sulphate15.76 g–Mordant
Distilled water1 L–Solvent
Haematein–1.876 gDye
Ethylene glycol, 50%, aqu.–1 LSolvent
MaterialHematal-8Hematal-16
Stock A1 vol2 vol
Stock B1 vol1 vol
Ethylene glycol, 50%, aqu.–1 vol
MaterialSolutionFunction
Standard AStandard B
Aluminum sulphate3.94 g–Mordant
Distilled water1 L–Solvent
Haematoxylin–5 gDye
Sodium iodate–0.5 gDye
Distilled water–1 LSolvent
Sulphuric acid, 0.5% aqueousas needed
Ammonia, 0.5% aqueousas needed

Compounding Procedures

Hematal-8 and Hematal-16 Solutions

  1. Prepare each stock solution separately.
  2. Combine as specified.
  3. The solution may be used immediately.

Standard Solution

  1. Prepare each solution separately.
  2. Standard solution B should be brought to a boil, then immediately cooled.

Protocol

Hematal-8 & Hematal-16 Solutions

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for the specified time.
    • Hematal-8 for 2-5 minutes.
    • Hematal-16 for 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.

Standard Solutions

  1. Bring sections to water with xylene and ethanol.
  2. Place into standard solution A for 1 hour.
  3. Rinse with water.
  4. Place into standard solution B for 20 minutes.
  5. Rinse with water.
  6. Place into 0.5% sulphuric acid for 40 seconds.
  7. Rinse with water.
  8. Place into 0.5% ammonia water for 5 seconds.
  9. Wash well with water.
  10. Optionally, counterstain with eosin.
  11. 70% ethanol for 30 seconds.
  12. 90% ethanol for 30 seconds.
  13. Absolute ethanol, 2 changes, 1 minute each.
  14. Xylene, 2 changes, 1 minute each.
  15. Mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • Hematal-8 and Hematal-16 may be used for routine staining. The solutions are quickly and conveniently prepared.
  • Hematal-8 may be used regressively by applying for 30 minutes, then lightly differentiating in acid. Dilute sulphuric acid was recommended (0.5% aqu.), but hydrochloric acid was usable.
  • Hematal-8 has 8 atoms of aluminum for each molecule of hematein, Hematal-16 has 16, hence the names.
  • 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.
  • The standard method was used to give consistent staining with the usually expected appearance, but with the ability to vary each step for experimental reasons. It was not intended for routine use.

Safety Note

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

References

  1. Baker, J. R., (1962),
    Experiments on the action of mordants: 2. Aluminium-haematein.
    Quarterly Journal of Microscopical Science, v. 103, pt. 4, pp. 493-517.
March 30, 2023
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Jones’ Impregnation for Basement Membranes

By Aldehydes, Carbohydrates, Metal Impregnation, Metal Impregnation, Silver, Protocols, Stain Target, Stain Type
Protocol

Jones' Impregnation

for Basement Membranes

13
steps
9
materials
print

Materials

  • Periodic acid, 0.5% aqu.
  • Neutral red, 1% aqu.
  • Yellow gold chloride, 0.2% aqu.
  • Light green SFy, 0.2% in 0.2% acetic acid, or progressive hemalum and eosin
  • Sodium thiosulfate, 2.5% aqu.
  • Stock Methenamine silver
    MaterialAmount
    Methenamine, 3% aqu.100mL
    Silver nitrate, 5% aqu.5mL

    Shake until the precipitate redissolves. Silvering of the container indicates deterioration.

  • Working Methenamine silver
    MaterialAmount
    Stock Methenamine silver50mL
    Borax, 5% aqu.5mL

    Make just before use and preheat to 50°C.

Tissue Sample

3µ paraffin sections of neutral buffered formalin or Bouin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended. Thinner sections are to be preferred. This method gives excellent results with deplasticized methyl methacrylate sections at 1µ.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Oxidize with 0.5% periodic acid for 15 minutes.
  3. Rinse well with tap water.
  4. Rinse with distilled water.
  5. Treat with methenamine silver solution at 50&degC. until impregnated (up to 3 hours)
  6. Wash with distilled water.
  7. Tone with 0.2% gold chloride solution for 2 minutes.
  8. Rinse with distilled water.
  9. Fix in 2.5% sodium thiosulfate for 3 minutes.
  10. Wash well with running tap water.
  11. Counterstain with light green, neutral red or a light H&E.
  12. Rinse with tap water.
  13. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Basement membranes  –  black
  • Oxidisable carbohydrates  –  black
  • Background  –  as counterstained

Notes

  • In order to see the basement membranes on edge it is necessary to use the thinnest sections possible, especially for glomeruli.
  • Methenamine is also known as hexamethylenetetramine and hexamine.
  • Borax is sodium tetraborate.
  • Toning is a variable step. Untoned sections give dark brown material on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black material on a pale grey-brown background. Others tone longer (a few minutes) to produce black material on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.
  • This method depends on a similar principle to the periodic acid Schiff reaction, but in which the aldehydes produced by oxidation reduce a silver solution instead of combining with Schiff’s reagent to form a red compound. Consequently, those materials which are red in a PAS will be black in Jones’ stain, i.e. it is not specific for basement membranes but will demonstrate any carbohydrates which can be oxidised to aldehydes.
  • Hayashi, Tome and Shimosato recommended that, after oxidation with periodic acid, thiosemicarbazide should be applied to the section. Thiosemicarbazide has the formula H2NNHCSNH2. The hydrazine group (H2NNH-) combines with any aldehydes generated by periodic acid oxidation. The thiocarbamyl group (-CSNH2) is a more powerful reducing agent than the aldehydes it replaces and reduces the methenamine silver solution more rapidly and with higher contrast.Immediately following step 3:
    • Place sections in 1% aqueous thiosemicarbazide for 10 minutes.
    • Wash well with tap water, and carry on from step 4.
  • It is well known that metallic azides can be explosive. However, thiosemicarbazide is not a simple metallic azide. The MSDS says:
    • Flash Point: n/a
    • Lower Explosive Limit: n/a
    • Upper Explosive Limit: n/a
    • Unusal Fire and Expl.rds: none identified

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.
  2. Hayashi, I., Tome, Y. and Shimosato, Y., (1989)
    Thiosemicarbazide used after periodic acid makes methenamine silver staining of renal glomerular basement membranes faster and cleaner.
    Stain Technology, v 64, p 185.
March 30, 2023
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Smith’s Vanadium Hematoxylin

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

Smith's Vanadium Hematoxylin

7
steps
5
materials
print

When differentiated with dilute lithium carbonate, this solution demonstrates basic proteins, including nuclear histones.

Materials

MaterialAmountFunction
Hematoxylin50 mgDye
Ethanol, 100%5 mLSolvent
Glycerol10 mLSolvent
Distilled water35 mLSolvent
Ammonium metavanadate200 mgSolvent

Compounding procedure

  1. Dissolve the hematoxylin in the ethanol, then add the glycerol and water in sequence.
  2. Add the ammonium vanadate and stir for 30 minutes.
  3. The ammonium vanadate may not completely dissolve. The pH is 6.2.

Tissue Sample

Paraffin sections of Schaudinn, Bouin or formalin fixed tissues are suitable. Sections of Zenker or Helly fixed tissues must be soaked in saturated aqueous lithium carbonate for 4 hours to stain satisfactorily.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the vanadate hematoxylin for 30 minutes.
  3. Place directly into 0.08% aqueous lithium carbonate for the appropriate time.
    Differentiation times in lithium carbonate

    FixativeTime
    Bouin2 minutes
    Formalin, 10%2 minutes
    Helly10 minutes
    Schaudinn5 minutes
    Zenker10 minutes
  4. Briefly rinse with distilled water for 2 seconds.
  5. Optionally, counterstain if wished.
  6. Dehydrate with 70%, 95% and absolute ethanols.
  7. Clear with xylene and mount with a resinous medium (Permount specified).

Expected Results

  • Nuclear histone  –  blue
  • Ribosomal protein  –  paler blue
  • Other protein  –  as counterstain or unstained

Notes

  • Counterstaining can be accomplished at step 5 with 1% aqueous eosin Y, phloxine B or erythrosin B.
  • Alternatively, metanil yellow at 0.1% in 95% ethanol containing 0.1% acetic acid may be used between the 70% and 95% ethanols in step 6.
  • Counterstaining with 0.01% aqueous safranin O stained RNA intensely, giving good contrast between blue nuclear histone and endoplasmic reticulum.

Safety Note

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

References

  1. Smith, A. A., (1995)
    A vanadate hematoxylin stain for basic proteins.
    Biotechnic and Histochemistry, v. 70, Nº 5, p. 5.
March 30, 2023
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HPS, AFIP Modification General Oversight stain

By Protocols, Stain Type, Trichrome Staining, Yellowsolve Staining
Protocol

HPS, AFIP Modification

General Oversight stain

11
steps
6
materials
print

Also known as the Hematoxylin Phloxine Saffron stain, AFIP modification.

Materials

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable, as are many other fixatives.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place in picric acid solution for 5 minutes.
  3. Wash with water to remove yellow.
  4. Stain nuclei with hemalum, differentiate and blue.
  5. Wash well with water.
  6. Place in solution A for 2 minutes.
  7. Wash with tap water for 5 minutes.
  8. Thoroughly dehydrate with absolute ethanol.
  9. Place into solution B for 5 minutes.
  10. Rinse well with absolute ethanol.
  11. Clear in xylene and mount with a synthetic resinous medium.

Expected Results

  • Nuclei  –  blue
  • Muscle & cytoplasm  –  red
  • Collagen  –  yellow

Notes

  • The saffron should be extracted by mixing with anhydrous ethanol in a tightly capped container, then placing in a 56°C oven for a few days. Store and use at room temperature. It has a limited life and is best when freshly made.
  • Saffron is expensive. It may be available in East Indian grocery stores or health food stores as its most common use today is as a spice and food coloring.
  • It is important that there be no water in the saffron solution and that sections be thoroughly dehydrated before it is applied.
  • Usually, whole stigmata are more effective than ground saffron.
  • Erythrosine B may be used instead of phloxine B.

Safety Note

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

References

  1. Histological Staining Methods of the Armed Forces Institute of Pathology, 3rd ed. (1968)
    Luna, Lee G.
    McGraw-Hill, NY, USA
March 30, 2023
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Akita & Kaneko’s Hemalum for Elastic Fibers

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

Akita & Kaneko's Hemalum

for Elastic Fibers

5
steps
6
materials
print

Materials

MaterialAmount
Hematoxylin100mg
Ethanol, 70%100mL
Sodium iodate20mg
Potassium alum300mg
Chloral hydrate5g
Citric acid100mg

Staining Solution Preparation

  1. Dissolve the hematoxylin into the ethanol.
  2. Add the potassium alum, then the other ingredients.
  3. Mix well for 15 to 20 minutes, then filter.
  4. At this point the solution is stable for 2 months at room temperature.
  5. Immediately prior to use, adjust to pH 8.0 with saturated aquueous lithium carbonate or 1N potassium hydroxide.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Helly and Bouin are also suitable. Other fixatives should be tested.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into freshly pH adjusted staining solution for 30-60 minutes.
  3. Wash with running tap water for 10 minutes.
  4. Dehydrate with ethanols.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  blue
  • Collagen  –  pale violet

Notes

  • Note that this is an ethanolic variant of Mayer’s hemalum.
  • If sodium iodate is left out, the staining solution lasts 3-4 days, but the staining time is extended to 60 minutes.
  • The solution may also be made by diluting 30 mL Mayer’s hemalum with 70 mL absolute ethanol, mixing for 20 minutes, and filtering before adjusting the pH. Staining requires 60 minutes and the results are paler.

Safety Note

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

References

  1. Akita, M. and Kaneko, K., (1981)
    An improved hematoxylin method with application of Mayer’s hemalum for staining of elastic fibres.
    Stain Technology, v. 56, p. 327
  2. Kaneko, K. and Akita, M., (1978)
    On the staining of elastic fibres with alum hematoxylin.
    Stain Technology, v. 53, p. 43.
March 30, 2023
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Gabe’s Aldehyde Fuchsin

By Aldehyde Fuchsin, Dye Type, Elastic Fibers, Protocols, Stain Target, Stain Type
Protocol

Gabe's Aldehyde Fuchsin

6
steps
9
materials
print

Materials

Stock powder

MaterialAmount
Pararosanilin5g
Paraldehyde5mL
Hydrochloric acid10mL
Distilled water1L

Dye Preparation Procedures

  1. Add the dye to the water and boil.
  2. Cool to room temperature.
  3. Add paraldehyde and hydrochloric acid.
  4. Ripen at RT two days. Filter. Wash precipitate with 50mL distilled water. Dry the ppt and paper at 60°C.
  5. Store in a labelled container. Stable for about ten years.

Working solution

MaterialAmount
Stock powder0.25g
Ethanol, 70%200mL
Acetic acid, glacial2mL

Acid ethanol

MaterialAmount
Hydrochloric acid, conc.1mL
Ethanol, 95%200mL

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 in the working solution for 5 minutes.
  3. Rinse well with running tap water.
  4. Rinse with acid ethanol for 20-30 seconds to remove excess dye.
  5. Counterstain if wished.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  purple
  • Mast cells  –  purple
  • Pituitary βcells  –  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.

Safety Note

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

References

  1. Kiernan. J.A., (1999)
    Histological and histochemical methods: Theory and practice, Ed. 3
    Butterworth Heinemann, Oxford, UK.
    Citing:
    Gabe, M., (1976)
    Histological techniques.
    Masson, Paris.
March 30, 2023
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