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Trichrome Staining

Bensley’s trichrome for Muscle and Collagen

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

Bensley's trichrome

for Muscle and Collagen

9
steps
7
materials
print

Materials

Solution A

MaterialAmount
Acid fuchsinTo saturation
Aniline water100mL

Solution Preparation

  1. Add about 20 g dye to the aniline water.
  2. Shake intermittently for 48 hours. Filter.

Solution B

MaterialAmount
Phosphomolybdic acid1g
Distilled water100mL

Solution C

MaterialAmount
Orange G2g
Aniline blue0.5g
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 into solution A for 10 minute.
  3. Rinse with distilled water.
  4. Place into solution B for 10 minutes.
  5. Rinse quickly with distilled water.
  6. Place into solution C for 60 minutes.
  7. Rinse with 95% ethanol until clouds of dye cease being extracted.
  8. Dehydrate with ethanol.
  9. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  red
  • Erythrocytes  –  orange
  • Cytoplasm  –  red
  • Collagen  –  blue

Notes

  • Solution A is Altmann’s acid fuchsin, originally formulated for staining mitochondria.
  • This method does not specify an acid resistant nuclear stain. Doing so before staining with solution A may improve the technique.

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:
    Mallory, F. B., (1938),
    Pathological technique.
    Saunders, Philadelphia, Pa, USA
March 30, 2023
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Bencosme’s HPS General Oversight stain

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

Bencosme's HPS

General Oversight stain

11
steps
5
materials
print

Also known as the Hematoxylin Phloxine Saffron stain.

Materials

  • Regressive hemalum
  • Solution A
    MaterialAmount
    Phloxine B2g
    Distilled water100mL

    Dissolve the phloxine B and filter. Preserve with 2-5 drops of strong formalin.

  • Solution B
    MaterialAmount
    Saffron6g
    Absolute ethanol200mL

    Mix the saffron stigmata with the ethanol and seal tightly. Place in an oven at 56°C for 1-2 weeks.

Tissue Sample

Brasil’s fixative was specified. 5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable, but results may be improved by refixing in Bouin’s fixative or saturated aqueous picric acid at 56°C for one hour prior to staining, then washing the sections in tap water to remove all yellow discoloration.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Treat with Bouin’s fluid if desired.
  3. Stain nuclei with hemalum, differentiate and blue.
  4. Wash well with water.
  5. Place in solution A for 2-20 minutes.
  6. Wash with tap water until red stain stops being removed, 1-5 minutes.
  7. Differentiate phloxine with 80% ethanol up to 1½ minutes.
  8. Thoroughly dehydrate with absolute ethanol.
  9. Place into solution B for 2-20 minutes until a suitable red-yellow balance is achieved.
  10. Rinse well with absolute ethanol.
  11. Clear with xylene and mount with a synthetic resinous medium.

Expected Results

  • Nuclei  –  blue
  • Cytoplasm  –  red shades
  • Muscle  –  pink
  • Elastic fibres  –  bright red
  • Collagen  –  orange-yellow

Notes

  • Any hemalum may be used, but Bencosme specified a particular formula.
  • 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. Usually, whole stigmata are more effective than ground saffron.
  • If the saffron solution is too strong it may be diluted with absolute ethanol. Store and use at room temperature. It has a limited life of about a month and is best when freshly made. It is important that there be no water in the saffron solution and that sections be thoroughly dehydrated before it is applied. If contaminated with moisture the solution must be discarded.

Safety Note

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

References

  1. Medical Laboratory Technology, 2nd ed. (1969)
    Lynch M.J., Raphael S.S., Mellor L.D., Spare P.D. and Inwood M.J.H.,
    W. B. Saunders Co., Toronto, On., Canada
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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Masson 44/41 for Fibrin

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

Masson 44/41

for Fibrin

12
steps
12
materials
print

With this trichrome stain full and complete fixation is absolutely essential. Minimalist formalin fixation and quick processing will give poorly stained erythrocytes with red or orange tinges instead of the yellow they should have. Lendrum and coworkers specified about seven days fixation in formal sublimate followed by processing thoroughly, sectioning, degreasing with trichlorethylene for 48 hours, then refixing in picro-mercuric-alcohol. Results are usually poor with formalin fixed material, even if treated with Bouin’s fluid at 56°C for an hour or so.

Materials

  • An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
  • Picro-mercuric ethanol
    MaterialAmount
    Ethanol, absolute100mL
    Picric acidto saturation
    Mercuric chlorideto saturation
  • Plasma stain
    MaterialAmount
    Ponceau 6R1g
    Acetic acid, glacial1mL
    Distilled water99mL
  • Fibre stain
    MaterialAmount
    Naphthalene blue black CS1g
    Acetic acid, glacial1mL
    Distilled water99mL
  • Polyacid
    MaterialAmount
    Phosphotungstic acid1g
    Distilled water100mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate for one week. They should be paraffin processed with a schedule that thoroughly and completely dehydrates, then thoroughly cleared with xylene and infiltrated with paraffin wax. This process would normally take 48 hours or longer. Err on the side of completeness, and do not attempt to shorten the process. Avoid rapid fixation and overnight processing, as this produces tissues that stain poorly. Sections should be 3-5 µ thick.

Protocol

  1. Dewax sections with xylene.
  2. Place into a sealed container of trichlorethylene at 56°C for 48 hours.
  3. Rinse well with absolute ethanol.
  4. Refix sections in picro-mercuric-ethanol for a minimum of 3 and preferably 24 hours.
  5. Remove mercury pigment with the iodine-thiosulphate sequence.
  6. Stain nuclei with an acid resistant nuclear stain.
  7. Place in the plasma stain for 5 minutes.
  8. Differentiate with the polyacid for 5 minutes.
  9. Place in the fibre stain for 30 minutes.
  10. Rinse briefly with 1% aqueous acetic acid.
  11. Dehydrate with ethanol.
  12. Clear with xylene and mount with a resinous medium.

Expected Results

  • Fresh fibrin  –  red
  • Older fibrin  –  black
  • Connective tissue  –  pale blue
  • Plasma cell inclusions  –  red

Notes

  • Ponceau 6R is also known as acid red 44.
  • Naphthalene blue black CS is also known as acid black 41.

Safety Note

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

References

  1. Lendrum, A. C., et. al. (1962)
    Studies on the character and staining of fibrin.
    Journal of clinical pathology, v. 15, p. 401.
March 30, 2023
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