Lillie's Trichrome

for Muscle and Collagen

steps

materials

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Materials

Weigert’s iron hematoxylin or equivalent
Bouin’ fluid
Solution A
Material Amount
Biebrich scarlet 0.5 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Solution B
Material Amount
Phosphomolybdic acid 2.5 g
Phosphotungstic acid 2.5 g
Distilled water 100 mL
Solution C
Material Amount
Fast green FCF 2.5 g
Acetic acid, glacial 2.5 mL
Distilled water 97.5 mL
Solution D
Material Amount
Acetic acid, glacial 1 mL
Distilled water 99 mL

Material	Amount
Biebrich scarlet	0.5	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Material	Amount
Phosphomolybdic acid	2.5	g
Phosphotungstic acid	2.5	g
Distilled water	100	mL

Material	Amount
Fast green FCF	2.5	g
Acetic acid, glacial	2.5	mL
Distilled water	97.5	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 Bouin’s fluid, formal sublimate or B5 fixation. If formalin is used, secondary fixation of sections with Bouin’s fluid at 56°C for an hour will improve staining.

Protocol

Bring sections to water via xylene and ethanol
1. If formalin fixed, refix in Bouins fluid for one hour at 56°C
2. Wash well in tap water to remove the yellow.
Stain nuclei with Weigert’s iron hematoxylin or equivalent.
Wash well in tap water, rinse with distilled water.
Place into solution A for 2 minutes.
Rinse with distilled water.
Place into solution B for 1 minute.
Rinse with distilled water.
Place into solution C for 2 minutes.
Place into solution D for 1 minute.
Dehydrate with acetone.
Clear with xylene and mount with a resinous medium

Expected Results

Nuceli – black
Cytoplasm – red
Muscle – red
Collagen – green

Notes

This method is frequently, and erroneously, called “Masson’s” trichrome.
Refixing the sections in Bouin’s fluid intensifies the colours and increases the contrast between the tissue components. Although often called mordanting, this step is simply secondary fixation of sections.
Acetone is specified for dehydration. Absolute ethanol can be substituted.
Longer times in solutions A and C (5 minutes each) may improve staining.

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.
Kiernan. J.A., (1999)
Histological and histochemical methods: Theory and practice, Ed. 3
Butterworth Heinemann, Oxford, UK.

March 30, 2023

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Mallory’s Trichrome for Muscle and Collagen

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

Protocol

Mallory's Trichrome

for Muscle and Collagen

steps

materials

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Materials

Solution A

Material	Amount
Acid fuchsin	1	g
Distilled water	100	mL

Solution B

Material	Amount
Phosphomolybdic acid	1	g
Distilled water	100	mL

Solution C

Material	Amount
Orange G	2	g
Methyl blue	0.5	g
Oxalic acid	2	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 2 minute.
Rinse with distilled water.
Place into solution B for 2 minutes.
Rinse quickly with distilled water.
Place into solution C for 15 minutes.
Wash well with distilled water.
Dehydrate and differentiate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – red
Erythrocytes – orange
Muscle – red
Collagen – blue

Notes

Phosphotungstic acid may be substituted for phosphomolybdic acid in solution B.
This is the method that introduced the concept, if not the name, of trichrome staining.

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, (1901)
Journal of Experimental Medicine, v.5, pp.15
And:
Mallory, (1936)
Stain technology, v.11, pp.101

March 30, 2023

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Masson’s Trichrome for Muscle and Collagen

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

Protocol

Masson's Trichrome

for Muscle and Collagen

steps

materials

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Materials

Weigert’s iron hematoxylin or equivalent.
Solution A
Material Amount
Acid fuchsin 0.5 g
Xylidine ponceau 0.5 g
Distilled water 99 mL
Glacial acetic acid 1 mL
Solution B
Material Amount
Phosphomolybdic acid 1 g
Distilled water 100 mL
Solution C
Material Amount
Light green SF yellowish 2 g
Glacial acetic acid 2 mL
Distilled water 100 mL

Material	Amount
Acid fuchsin	0.5	g
Xylidine ponceau	0.5	g
Distilled water	99	mL
Glacial acetic acid	1	mL

Material	Amount
Light green SF yellowish	2	g
Glacial acetic acid	2	mL
Distilled water	100	mL

Tissue Sample

5 µ paraffin sections of Bouin, formal sublimate or B5 fixed tissues are preferred. Zenker and Helly fixation is usually satisfactory. Formalin variants are generally adequate, but staining can be improved by secondary fixation of cut sections with Bouin’s fluid.

Protocol

Bring sections to water via xylene and ethanol.
1. If formalin fixed, refix in Bouins fluid for one hour at 56°C
2. Wash well in tap water to remove the yellow colour.
Stain nuclei with Weigert’s iron hematoxylin or equivalent.
Wash well in tap water, rinse with distilled water.
Place into solution A for 10 minutes.
Rinse with distilled water.
Place into solution B for 5 minutes.
Rinse with distilled water.
Place into solution C for 10 minutes.
Rinse well with distilled water.
Dehydrate with Ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Cytoplasm – red
Erythrocytes – red
Muscle – red
Collagen – green
Nuclei – dark brown

Notes

Since Masson gave details of his original method, there have been numerous variations published. One common variant is Lillie’s, a method often erroneously called “Masson’s” trichrome. These variants differ in the specifics of the dyes used, the concentration of the dyes and polyacid and the times for which they are applied.
The times given here should be considered a guide, although they will generally be found satisfactory if the procedure is followed completely, including refixation of sections in Bouin’s fluid.
Refixing the sections in Bouin’s fluid intensifies the colours and increases the contrast between the tissue components. It is sometimes incorrectly referred to as mordanting, but it is simply a form of secondary fixation of sections and has no real mordanting effect. It should be noted that primary fixation in Bouin’s fluid is recommended for this method.
The dyes in solution A stain the muscle, fibrin, cytoplasm and erythrocytes and the solution is sometimes referred to as the plasma stain. Acid fuchsin and xylidine ponceau are the dyes originally recommended. Many other red acid dyes have been suggested. Biebrich scarlet, for instance, being used by Lillie.
Solution C is sometimes called the fibre stain. It is used to colour collagen. Some other materials will also be coloured, but the method is not used to demonstrate them. Light green SF yellowish is commonly used, but fast green FCF gives a more emerald green. This is preferred by some microscopists. Although this latter dye is considered less likely to fade, it is not as commonly used.
Aniline blue, or either of its constituents, methyl blue, or water blue may be substituted alone or in combination if blue stained collagen is preferred to green.

Safety Note

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

References

The method described is in common use in many laboratories. A typical description would be as found in:

Theory and practice of histological techniques,
Bancroft, J. D. and Stevens, A.
Churchill Livingstone, London, England

March 30, 2023

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Masson’s Trichrome Original Variants

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

Protocol

Masson's Trichrome

Original Variants

steps

materials

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Expected Results

massons-trichrome-staining-rat-airway-tissue-600x400

Figure 1. Masson's Trichrome Staining of Rat Airway Tissue

Masson's trichrome stain of a paraffin embedded rat airway section with nuclei (in dark red/purple), cytoplasm (in red/pink), and connective tissue (in blue). Masson's trichrome stain (rat airway section) by Pierre Camateros is licensed under CC BY-SA 2.0.

Nuclei – Black (red if an iron hematoxylin was not used)
Collagen – blue
Bone – dark blue
Epithelia – light red
Muscle – light red
Erythrocytes – orange
Nervous tissue – violet

Materials

Régaud’s iron hematoxylin or equivalent
Solution A
Material Var 1 Var 2 Var 3 Var 4
Acid fuchsin 0.5 g 0.35 g – 1.0 g
Ponceau 2R – 0.65 g 1.0 g 1.0 g
Glacial acetic acid 0.5 mL 1 mL 1 mL 1 mL
Distilled water 100 mL 100 mL 100 mL 100 mL
Solution B
Material Amount
Phosphomolybdic acid 1 g
Distilled water 100 mL
Solution C
Material Amount
Acetic acid, glacial 2.5 mL
Distilled water 97.5 mL
Aniline blue as required
Mix the acetic acid and water. Saturate with aniline blue.
Solution D
Material Amount
Acetic acid, glacial 1 mL
Distilled water 99 mL
Solution E
Material Amount
Acetic acid, glacial 1 mL
Ethanol, absolute 99 mL

Material	Var 1	Var 2	Var 3	Var 4
Acid fuchsin	0.5	g	0.35	g	–		1.0	g
Ponceau 2R	–		0.65	g	1.0	g	1.0	g
Glacial acetic acid	0.5	mL	1	mL	1	mL	1	mL
Distilled water	100	mL	100	mL	100	mL	100	mL

Material	Amount
Acetic acid, glacial	2.5	mL
Distilled water	97.5	mL
Aniline blue	as required

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.
Optionally, stain nuclei with Régaud’s iron hematoxylin.
Wash in water.
Place into one of the variants of solution A for 5 minutes.
Rinse quickly with water.
Place into solution B for 5 minutes.
Drain, but do not rinse with water.
Place into solution C for 2-5 minutes.
Place into solution D until differentiated.
Dehydrate with solution E.
Clear with salicylic xylene.
Mount with salicylic balsam.

Notes

Masson also suggested another method.
The dye Masson called ponceau 2R is thought to be xylidine ponceau.
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, then allowing the excess to settle out. The medium should be clear, pale yellow. An 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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Masson’s Trichrome An Original Variant

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

Protocol

Masson's Trichrome

An Original Variant

steps

materials

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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

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

steps

materials

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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
Metanil yellow	to	saturation
Distilled water	100	mL

Tissue Sample

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

steps

materials

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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

steps

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

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

steps

materials

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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

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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Double Oxidation Schiff

By Carmen WongAldehydes, Periodic Acid-Schiff Reaction, Protocols, Schiff's Reagent Reactions, Stain Target, Stain Type

Protocol

Double Oxidation Schiff

steps

materials

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Double oxidation may be used with any procedure which demonstrates aldehydes, including Schiff’s reagent and methenamine silver reduction. Its use is not necessarily confined to staining fungi and it may be found useful when demonstrating other structures and the background stains too darkly.

Materials

Periodic acid – 1% aqueous
Analine-acetic block
Material Amount
Acetic acid, glacial 5 mL
Aniline oil 45 mL

Material	Amount
Acetic acid, glacial	5	mL
Aniline oil	45	mL

Tissue Sample

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

Protocol

Bring sections to water via xylene and ethanol.
Oxidize with periodic acid for 10 minutes.
Rinse well with tap water.
Treat with aniline-acetic for 30 minutes.
Rinse well with tap water.
Re-oxidize with periodic acid for 20 minutes.
Rinse well with tap water.
Rinse with distilled water.
Place in Schiff’s reagent for 10-30 minutes.
Wash off with distilled water.
Wash well with tap water for about 10 minutes.
Counterstain with Mayer’s hemalum for 2 minutes.
Wash well with tap water until hemalum is blued.
Dehydrate with ethanol, clear with xylene and coverslip using a resinous medium.

Expected Results

Oxidizable carbohydrates – red
Fungi – red
Nuclei & background – blue

Notes

The time periodic acid is applied in the first oxidation determines the amount of background staining eliminated. If there is only a small amount of this material, then all of it may be blocked
The first oxidation will also oxidize some of the target material, and this staining will also be inhibited. If the first oxidation is applied for too long, the depth of staining of the target material may be affected. The 10 minutes specified is usually enough.
The time periodic acid is applied in the second oxidation determines the depth of staining for the target material. 20 minutes is usually adequate, but extending it may give darker staining. At some point, extending the time in periodic acid will cease to produce darker staining as all available carbohydrate will have been oxidized.

Safety Note

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

References

Personal observation, B. Llewellyn,

March 30, 2023

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