Masson's Trichrome

An Original Variant

13

steps

6

materials

print

Materials

Solution A

Material	Amount
Hematoxylin	1	g
Distilled water	100	mL

Solution B

Material	Amount
Ferric ammonium sulphate	4	g
Distilled water	100	mL

Solution C

Material	Amount
Ferric ammonium sulphate	2	g
Distilled water	100	mL

Solution D

Material	Amount
Acid fuchsin	0.1	g
Distilled water	100	mL

Solution E

Material	Amount
Phosphomolybdic acid	1	g
Distilled water	100	mL

Solution F

Material	Amount
Aniline blue	0.5	g
Phosphomolybdic acid	0.5	g
Distilled water	100	mL

Dissolve separately and combine.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Trichrome stains often benefit from initial Bouin or formal sublimate fixation, or from secondary fixation in Bouin’s fluid for an hour at 56°C.

Protocol

Bring sections to water via xylene and ethanol.
Place into solution A for 5 minutes at 50°C.
Rinse rapidly with water at 50°C.
Place into solution B for 10-15 minutes at 50°C.
Differentiate in solution C until nuclei alone are stained.
Wash in running tap water for 15 minutes.
Place into solution D for 10 minutes.
If overstained, remove excess with tap water.
Place in solution E for 5-10 minutes.
Place in solution F for 20-60 minutes.
Rinse rapidly with water.
Dehydrate with 95% and absolute ethanols.
Clear with xylene and mount with a resinous medium.

Expected Results

Muscle – red
Collagen – blue
Nuclei – black

Notes

This method was published at the same time as Mason’s more commonly used technique.
Steps 1-6 are an acid resistant nuclear stain. Another could be substituted.

Safety Note

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

References

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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Masson’s Trichrome Yellow Collagen Variant

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Masson's Trichrome

Yellow Collagen Variant

12

steps

6

materials

print

Materials

Régaud’s iron hematoxylin or equivalent
Solution A
Material Amount
Acid fuchsin 1 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Solution B
Material Amount
Phosphomolybdic acid 1 g
Distilled water 100 mL
Solution C
Material Amount
Metanil yellow to saturation
Distilled water 100 mL
Solution D
Material Amount
Acetic acid, glacial 1 mL
Distilled water 99 mL

Material	Amount
Acid fuchsin	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Material	Amount
Metanil yellow	to	saturation
Distilled water	100	mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Trichrome stains often benefit from initial Bouin or formal sublimate fixation, or from secondary fixation in Bouin’s fluid for an hour at 56°C.

Protocol

Bring sections to water via xylene and ethanol.
Stain nuclei with Régaud’s iron hematoxylin or equivalent.
Wash well with water.
Place into solution A for 5 minutes.
Rinse rapidly with water.
Place into solution B for 5 minutes.
Drain.
Pour on solution C and leave 5 minutes.
Place in solution D for 5 minutes.
Rinse rapidly with water.
Dehydrate with absolute ethanol.
Clear with salicylic xylene and mount with salicylic balsam.

Expected Results

Nuclei – black
Muscle – red
Collagen – yellow

Notes

This method was published at the same time as Mason’s more commonly used technique.
It was specified that solution C should be poured onto the slide while on a staining rack, probably to ensure the solution was not re-used.
It was originally considered important to mount sections stained with acid dyes in an acidified mounting medium, in the belief that it preserved brightness and clarity.
Salicylic xylene is made by saturating xylene with salicylic acid.
Salicylic balsam is made by adding salicylic acid to Canada balsam, allowing any excess to clear by settling out. A useful alternative is to dip the coverslip into salicylic xylene prior to mounting. The dissolved salicylic acid will then acidify the mounting medium.

Safety Note

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

References

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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Milligan’s trichrome for Muscle and Collagen

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Milligan's trichrome

for Muscle and Collagen

14

steps

9

materials

print

Materials

Solution A

Material	Amount
Potassium dichromate	2.25	g
Hydrochloric acid, conc	2.25	mL
Ethanol, 95%	25	mL
Distilled water	75	mL

Solution B

Material	Amount
Acid fuchsin	0.1	g
Distilled water	100	mL

Solution C

Material	Amount
Phosphomolybdic acid	1	g
Distilled water	100	mL

Solution D

Material	Amount
Orange G	2	g
Phosphomolybdic acid	1	g
Distilled water	100	mL

Solution E

Material	Amount
Acetic acid, glacial	1	mL
Distilled water	100	mL
Directions

Solution F

Material	Amount
Fast green FCF	0.1	g
Acetic acid, glacial	0.2	mL
Distilled water	100	mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed were specified. Other fixatives may 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

Bring sections to water via xylene and ethanol.
Place into solution A for 5 minute.
Rinse with distilled water.
Place into solution B for 5 minutes.
Rinse quickly with distilled water.
Place into solution C for 1-5 minutes.
Rinse with distilled water.
Place into solution D for 5-10 minutes.
Rinse with distilled water.
Place into solution E for 2 minutes.
Place into solution F for 5-10 minutes.
Place into solution E for 3 minutes.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – red
Erythrocytes – orange
Muscle – red
Collagen – green

Notes

Aniline blue may be substituted for fast green FCF at the same concentration. Collagen would then be blue.

Safety Note

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

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Milligan, (1946)
Technical Bulletin, v. 7, pp.57

March 30, 2023

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Möllendorf’s trichrome for Muscle and Collagen

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Möllendorf's trichrome

for Muscle and Collagen

13

steps

6

materials

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Materials

Hansen’s iron hematoxylin or equivalent
Solution A
Material Amount
Eosin Y 1 g
Acetic acid, glacial 0.3 mL
Distilled water 100 mL
Solution B
Material Amount
Phosphomolybdic acid 2 g
Distilled water 100 mL
Solution C
Material Amount
Methyl blue 1 g
Distilled water 100 mL

Material	Amount
Eosin Y	1	g
Acetic acid, glacial	0.3	mL
Distilled water	100	mL

Material	Amount
Phosphomolybdic acid	2	g
Distilled water	100	mL

Material	Amount
Methyl blue	1	g
Distilled water	100	mL

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

Bring sections to water via xylene and ethanol.
Stain nuclei with Hansen’s iron hematoxylin for 5 minutes.
Rinse with distilled water.
Wash with running tap water.
Place into solution A for 20 minute.
Rinse with distilled water.
Place into solution B for 10 seconds.
Rinse with distilled water.
Place into solution C for 1-2 minutes.
Rinse with distilled water.
Place into 95% ethanol until clouds of dye cease to flood away.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – black
Cytoplasm – pink
Muscle – pink
Collage – blue

Safety Note

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

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Roskin, G.E., (1946)
Mikroskopecheskaya technika, pp.158
Moscow, Sovetskaya Nauka.

March 30, 2023

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Patay’s Trichrome for Muscle and Collagen

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Patay's Trichrome

for Muscle and Collagen

9

steps

6

materials

print

Materials

Masson’s alum hematoxylin
Solution A
Material Amount
Ponceau 2R 1 g
Distilled water 100 mL
Solution B
Material Amount
Phosphomolybdic acid 1 g
Distilled water 100 mL
Solution C
Material Amount
Light green SF 0.5 g
Ethanol, 90% 100 mL

Material	Amount
Ponceau 2R	1	g
Distilled water	100	mL

Material	Amount
Light green SF	0.5	g
Ethanol, 90%	100	mL

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

Bring sections to water via xylene and ethanol.
Stain nuclei with Masson’s alum hematoxylin, underdifferentiate and blue.
Place into solution A for 2 minute.
Rinse with distilled water.
Place into solution B for 2 minutes.
Rinse quickly with distilled water.
Place into solution C for 30 seconds.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Cartilage – blue
Erythrocytes – yellow
Cytoplasm – orange
Muscle – orange
Bone – bright green
Collagen – green

Notes

Gray strongly recommended this technique. He considered it to be the finest trichrome described to that date (1954).
The blue of the cartilage is from using a ripened, strong hemalum.

Safety Note

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

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Patay, (1934)
Bulletin d’histologie appliquée à la physologie et à la pathologie et de technique microscopique, v.11, pp.408

March 30, 2023

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Mollier’s trichrome for Elastic and Collagen

By Carmen WongDye Type, Elastic Fibers, Orcein, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Mollier's trichrome

for Elastic and Collagen

13

steps

13

materials

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Materials

Weigert’s iron hematoxylin or equivalent
Solution A
Material Amount
Orcein 0.8 g
Hydrochloric acid 1 mL
Ethanol, 100% 50 mL
Distilled water 50 mL
Solution B
Material Amount
Azocarmine 2 g
Acetic acid, glacial 1 mL
Distilled water 100 mL
Solution C
Material Amount
Phosphotungstic acid 5 g
Distilled water 100 mL
Solution D
Material Amount
Naphthol green B 1 g
Acetic acid, glacial 1 mL
Distilled water 100 mL

Material	Amount
Orcein	0.8	g
Hydrochloric acid	1	mL
Ethanol, 100%	50	mL
Distilled water	50	mL

Material	Amount
Azocarmine	2	g
Acetic acid, glacial	1	mL
Distilled water	100	mL

Material	Amount
Naphthol green B	1	g
Acetic acid, glacial	1	mL
Distilled water	100	mL

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

Bring sections to water via xylene and ethanol.
Place into solution A for 12 hours.
Stain nuclei with Weigert’s iron hematoxylin for 1-3 minutes.
Differentiate the nuclear stain if necessary.
Wash with water for 15 minutes.
Place into solution B for 15-30 minutes.
Rinse with distilled water.
decolorise in solution C for 2-6 hours, changing the solution three times.
Rinse quickly with distilled water.
Place into solution D for 15-30 minutes.
Agitate vigorously in 95% ethanol for 30 seconds.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Elastic – black
Erythrocytes – red
Cytoplasm – purple
Collagen – green

Notes

Solution A is the Unna-Taenzer elastic solution.

Safety Note

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

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Mollier, (1938)
Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik, v.55, pp.472.
And:
von Kahlden, C. and Laurent, O., (1896)
Technique microscopique, pp.143
Carré, Paris, France

March 30, 2023

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Kohashi’s Trichrome for Elastic and Collagen

By Carmen WongDye Type, Elastic Fibers, Orcein, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Kohashi's Trichrome

for Elastic and Collagen

12

steps

14

materials

print

Materials

Solution A

Material	Amount
Azocarmine	0.1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Solution B

Material	Amount
Aniline	0.1	mL
Ethanol, 95%	100	mL

Solution C

Material	Amount
Acetic acid, glacial	1	mL
Ethanol, 95%	100	mL

Solution D

Material	Amount
Phosphotungstic acid	5	g
Distilled water	100	mL

Solution E

Material	Amount
Eosin B	0.7	g
Acid fuchsin, sat. aqu.	4	mL
Unna’s elastic stain	35	mL
Ethanol, 50%	35	mL
Glycerol	40	mL

Tissue Sample

Many fixatives are satisfactory. 5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. 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

Bring sections to water via xylene and ethanol.
Place into solution A for 12-15 minutes.
Rinse quickly with distilled water.
Differentiate nuclei with solution B.
Place into solution C for 30-60 seconds.
Rinse with distilled water.
Place into solution D for 30-60 minutes.
Rinse with distilled water.
Place into solution E for 15-20 minutes.
Place into 95% ethanol until differentiated.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – red
Erythrocytes – orange
Elastic fibres – purple
Cytoplasm – red
Collagen – blue

Notes

Solution E is Pasini’s trichrome solution.

Safety Note

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

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Kohashi, (1937)
Folia anatomica Japonica, v.15, pp.175

March 30, 2023

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Brillmeyer’s Trichrome for Muscle and Collagen

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Brillmeyer's Trichrome

for Muscle and Collagen

8

steps

6

materials

print

Materials

Solution A

Material	Amount
Acid fuchsin	0.2	g
Distilled water	100	mL

Solution B

Material	Amount
Aniline blue	0.5	g
Orange G	2	g
Phosphomolybdic acid	1	g
Distilled water	100	mL

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.

Protocol

Bring sections to water via xylene and ethanol.
Stain with an acid resistant nuclear stain.
Place into solution A for 1 minute.
Drain.
Place into solution B for 2-3 hours.
Wash briefly with distilled water.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue or black
Erythrocytes – yellow
Cytoplasm – red
Collagen – blue

Notes

The original technique recommended nuclear staining in Delafield’s alum hematoxylin, with blueing.
Weiss modified this method to shorten the time required.

Safety Note

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

References

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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Bensley’s trichrome for Muscle and Collagen

By Carmen WongProtocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Bensley's trichrome

for Muscle and Collagen

9

steps

7

materials

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Materials

Solution A

Material	Amount
Acid fuchsin	To saturation
Aniline water	100	mL

Solution Preparation

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

Solution B

Material	Amount
Phosphomolybdic acid	1	g
Distilled water	100	mL

Solution C

Material	Amount
Orange G	2	g
Aniline blue	0.5	g
Distilled water	100	mL

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

Bring sections to water via xylene and ethanol.
Place into solution A for 10 minute.
Rinse with distilled water.
Place into solution B for 10 minutes.
Rinse quickly with distilled water.
Place into solution C for 60 minutes.
Rinse with 95% ethanol until clouds of dye cease being extracted.
Dehydrate with ethanol.
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

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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Masson 44/41 for Fibrin

By Carmen WongFibrin, 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
Material Amount
Ethanol, absolute 100 mL
Picric acid to saturation
Mercuric chloride to saturation
Plasma stain
Material Amount
Ponceau 6R 1 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Fibre stain
Material Amount
Naphthalene blue black CS 1 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Polyacid
Material Amount
Phosphotungstic acid 1 g
Distilled water 100 mL

Material	Amount
Ethanol, absolute	100	mL
Picric acid	to saturation
Mercuric chloride	to saturation

Material	Amount
Ponceau 6R	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Material	Amount
Naphthalene blue black CS	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Material	Amount
Phosphotungstic acid	1	g
Distilled water	100	mL

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

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

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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