Paquin & Goddard's Trichrome

for Elastic and Collagen

steps

materials

print

Materials

Paquin & Goddard’s iron hematoxylin or equivalent
Solution A
Material Amount
Eosin Y 0.7 g
Phloxine 0.3 g
Orange G 0.1 g
Phosphotungstic acid 1 g
Distilled water 1 L
Solution B
Material Amount
Phosphotungstic acid 2 g
Distilled water 1 L
Solution C
Material Amount
Acetic acid, glacial 4 mL
Distilled water 1 L
Solution D
Material Amount
Aniline blue 0.4 g
Acetic acid, glacial 10 mL
Distilled water 990 mL

Material	Amount
Eosin Y	0.7	g
Phloxine	0.3	g
Orange G	0.1	g
Phosphotungstic acid	1	g
Distilled water	1	L

Material	Amount
Acetic acid, glacial	4	mL
Distilled water	1	L

Material	Amount
Aniline blue	0.4	g
Acetic acid, glacial	10	mL
Distilled water	990	mL

Tissue Sample

5µ paraffin sections of Masson’s picric-chrome-formalin fixation was specified. Most trichrome stains benefit from picric acid or mercuric chloride fixation. The specified fixation may be compensated for by secondary fixation of sections in Bouin’s fluid.

Masson’s picric-chrome-formalin contains:

Material	Amount
Picric acid, sat. aqu.	187.5	mL
Chromium aluminum sulphate	7.5	g
Formalin, Conc. 40%	67.5	mL

Add the alum to the formalin and leave for 1 hour.
Add the picric acid solution.
Let stand for 24 hours, and filter.
Return to tissue sample.

Protocol

Bring sections to water via xylene and ethanol.
Stain with Paquin & Goddard’s iron hematoxylin.
Wash with water.
Place into solution A for 5 minutes.
Place into solution B for 5 minutes.
Rinse twice with solution C.
Place into solution D for 5 minutes.
Rinse twice with solution C.
Place into solution B for 5 minutes.
Place into solution C for 30 minutes.
Rinse quickly with 95% ethanol.
Dehydrate with iso-amyl alcohol.
Clear with toluene and mount with a resinous medium.

Expected Results

Nuclei – black
Elastic – red
Cytoplasm – pink
Collagen – blue

Notes

If iso-amyl alcohol is not available, t-butanol is often suitable when the staining is likely to be removed with ethanol, although rapid dehydration with ethanol can often overcome this.
Although toluene was specified for clearing, xylene is likely suitable.

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:
Paquin & Goddard, (1947)
Bulletin of the International Association of Medical Museums and
Journal of Technical Methods, v.27, pp.198

March 30, 2023

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Pasini’s Trichrome for Elastic and Collagen

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

Protocol

Pasini's Trichrome

for Elastic and Collagen

steps

materials

print

Materials

Ehrlich’s alum hematoxylin
Solution A
Material Amount
Phosphotungstic acid 2 g
Distilled water 100 mL
Solution B – Var I
Material Amount
Eosin B 0.7 g
Acid fuchsin, sat. aqu. 4 mL
Unna’s elastic stain 35 mL
Glycerol 40 mL
Ethanol, 50% 35 mL
Solution B – Var II
Material Amount
Eosin B, 2% in 50% ethanol 30 mL
Acid fuchsin, sat. aqu. 4 mL
Unna’s elastic stain 30 mL
Glycerol 15 mL

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

Material	Amount
Eosin B, 2% in 50% ethanol	30	mL
Acid fuchsin, sat. aqu.	4	mL
Unna’s elastic stain	30	mL
Glycerol	15	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 an alum hematoxylin nuclear stain.
Place into solution A for 10 minute.
Rinse quickly with distilled water.
Place into solution B for 15-20 minutes.
Rinse quickly with 70% ethanol.
Dehydrate and differentiate with 100% ethanol for a few seconds.
Clear with xylene and mount with a resinous medium.

Expected Results

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

Notes

Solution B var I was given by Gray, var II was given by Gatenby & Beams.

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:
Gatenby, J. B. & Cowdry, E. V., (1928)
Bolles Lee’s Microtomist’s Vade Mecum, pp. 280
Blakiston, Philadelphia
Bolles Lee, A.. Edited by Gatenby, J.B. and Beams, H.W., (1950)
The Microtomist’s Vade-Mecum. 11 ed., pp. 485
Churchill, London, UK.

March 30, 2023

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Garvey’s Stain for Elastic, Fibrin & Collagen

By Carmen WongDye Type, Elastic Fibers, Fibrin, Iron Hematoxylin, Protocols, Stain Target, Stain Type

Protocol

Garvey's Stain

for Elastic, Fibrin & Collagen

steps

materials

print

Materials

Stock Verhoeff A

Material	Amount
Hematoxylin	3	g
Ethanol, absolute	100	mL

Stock Verhoeff B

Material	Amount
Ferric chloride	2.3	g
Distilled water	100	mL

Stock Verhoeff C

Material	Amount
Potassium iodide	4	g
Iodine	2	g
Distilled water	100	mL

Working Verhoeff

Material	Amount
Stock solution A	3	volumes
Stock solution B	2	volumes
Stock solution C	1	volume

Polyacid

Material	Amount
Phosphotungstic acid	2	g
Distilled water	100	mL

Acetic water

Material	Amount
Acetic acid, glacial	0.5	mL
Distilled water	100	mL

Picro-mercuric chloride

Material	Amount
Picric acid, sat. aqu.	100	mL
Mercuric chloride	5	g
Distilled water	100	mL

Erythrocyte stain

Material	Amount
Lissamine fast yellow	1	g
Acetic acid, glacial	0.5	mL
Distilled water	100	mL

Plasma stain

Material	Amount
Biebrich scarlet	0.75	g
Acid fuchsin	0.75	g
Ponceau 2R	0.75	g
Acetic acid, glacial	0.5	mL
Distilled water	100	mL

Material	Amount for Var I		Amount for Var II
Aniline blue	0.5	g	–
Light green SF yellowish	–		2	g
Acetic acid, glacial	1	mL	2	mL
Distilled water	100	mL	100	mL

Tissue Sample

Formol sublimate fixation is preferred, although 10% formalin variants are acceptable. Paraffin sections at 3µ are preferred.

Protocol

Bring sections to water via xylene and ethanol.
1. Optionally, place into picro-mercuric chloride for 1 hour.
2. Wash well with warm water to remove picric acid.
Place in verhoeff’s solution for 9 minutes.
Wash with warm tap water for 5 minutes.
Place in lissamine fast yellow for 2 minutes.
Rinse with 0.5% acetic acid.
Place in the plasma stain for 5 minutes.
Rinse with distilled water.
Place in polyacid for 10 minutes.
Rinse with distilled water.
Place in a fiber stain variant for 2 minutes.
Rinse with 0.5% acetic acid.
Dehydrate, clear and mount with a resinous medium.

Expected Results

Nuclei – black
Elastic fibres – black
Fibrin – young – yellow
Fibrin – mature – red
Fibrin – old – blue or green
Erythrocytes – yellow
Muscle – red
Collagen – blue or green (some may be red)

Notes

Sections should be treated with picro-mercuric chloride if formol sublimate or similar fixation was not used initially.
The ferric chloride should be the hexahydrate crystals.
Although the iodine content of the Verhoeff elastic stain component may remove any mercury pigment, in methods such as this, some technologists prefer to apply the iodine-thiosulphate sequence before staining.

Safety Note

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

References

Garvey W., et. al., (1987),
A combined elastic, fibrin and collagen stain
Stain Technology, V. 62, No 6, pp 365-367.

March 30, 2023

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Garvey-Movat Pentachrome for Elastic, Mucin & Collagen

By Carmen WongDye Type, Elastic Fibers, Iron Hematoxylin, Protocols, Stain Target, Stain Type

Protocol

Garvey-Movat Pentachrome

for Elastic, Mucin & Collagen

steps

materials

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Expected Results

Movat staining showing amyloid, lipofuscin, and myocardial fibrosis in an autopsy of senile cardiac amyloidosis

fig1-movat-staining-amyloid-lipofuscin-myocardial-fibrosis-autopsy-senile-cardiac-amyloidosis-600x400

Figure 1. Movat Staining Showing Elastic Fibres in an Autopsy Specimen of Senile Cardiac Amyloidosis

Movat staining of an autopsy specimen of senile cardiac amyloidosis shows nuclei and elastic fibres (in black), collagen and reticular fibers (in yellow), ground substance and mucin (in blue), fibrin (in bright red), and muscle (in red). Amyloid is visible as extracellular muddy brown material (right of image), abundant lipofuscin as dark red granular material, and myocardial fibrosis as yellow (left of image). Cardiac amyloidosis very high mag movat by Nephron / Michael Bonert, Pathology & Molecular Medicine, McMaster University is licensed under CC BY-SA 3.0.

Nuclei – black
Elastic fibres – black
Fibrin – mature – red
Muscle – red
Collagen – yellow
Ground substance – blue-green

Materials

Stock Verhoeff A

Material	Amount
Hematoxylin	3	g
Ethanol, absolute	100	mL

Stock Verhoeff B

Material	Amount
Ferric chloride	2.3	g
Distilled water	100	mL

Stock Verhoeff C

Material	Amount
Potassium iodide	4	g
Iodine	2	g
Distilled water	100	mL

Working Verhoeff

Material	Amount
Stock solution A	3	volumes
Stock solution B	2	volumes
Stock solution C	1	volume

Polyacid

Material	Amount
Phosphotungstic acid	2	g
Distilled water	100	mL

Acetic water

Material	Amount
Acetic acid, glacial	0.5	mL
Distilled water	100	mL

Picro-mercuric chloride

Material	Amount
Picric acid, sat. aqu.	100	mL
Mercuric chloride	5	g
Distilled water	100	mL

Alcian blue

Material	Amount
Alcian blue	1	g
Acetic acid, glacial	3	mL
Distilled water	100	mL

Plasma stain

Material	Amount
Biebrich scarlet, 1% aqu.	8	mL
Acid fuchsin, 1% aqu.	2	mL
Acetic acid, 1% aqu.	100	mL

Fibre stain

Material	Amount
Saffron du Gatinais	6	g
Ethanol, absolute	100	mL

Preparation

Add the saffron to the ethanol and seal the container.
Incubate at 56°C for 2 weeks. This solution must be anhydrous.

Tissue Sample

Formol sublimate fixation is preferred, although 10% formalin variants are acceptable. Paraffin sections at 3µ are preferred.

Protocol

Bring sections to water via xylene and ethanol.
1. Optionally, place into picro-mercuric chloride for 1 hour.
2. Wash well with warm water to remove picric acid.
Rinse with 3% acetic acid.
Place in alcian blue at 60°C for 10 minutes
Rinse with distilled water.
Place in working Verhoeff’s solution for 6 minutes.
Wash with warm tap water for 6 minutes.
Place in the plasma stain for 3 minutes
Rinse with distilled water.
Place in the polyacid for 15 minutes.
Rinse with 1% acetic acid.
Dehydrate thoroughly with absolute ethanol 3 changes.
Place in the fiber stain for 5-6 minutes.
Dehydrate, clear and mount with a resinous medium

Notes

Sections should be treated with picro-mercuric chloride if formol sublimate or similar fixation was not used initially.
The ferric chloride should be the hexahydrate crystals.
Sections thicker than 4µ may need the elastic stain differentiated by treating with 1% aqueous ferric chloride for 20-30 seconds, then washing well with tap water.
Although the iodine content of the Verhoeff elastic stain component may remove any mercury pigment, in methods such as this, some technologists prefer to apply the iodine-thiosulphate sequence before staining.

Safety Note

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

References

Garvey W., et. al., (1986),
Improved Movat pentachrome stain
Stain Technology, V. 61, No 1, pp 60-62.

March 30, 2023

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Silverman-Movat Pentachrome for Elastic, Mucin & Collagen

By Carmen WongDye Type, Elastic Fibers, Orcein, Protocols, Stain Target, Stain Type

Protocol

Silverman-Movat Pentachrome

for Elastic, Mucin & Collagen

steps

materials

print

Expected Results

fig1-movat-staining-amyloid-lipofuscin-myocardial-fibrosis-autopsy-senile-cardiac-amyloidosis-600x400

Figure 1. Movat Staining Showing Elastic Fibres in an Autopsy Specimen of Senile Cardiac Amyloidosis

Nuclei – dark purple to black
Elastic fibres – purple to black
Muscle – Red, with longitudinal myofibrils, cross striations and intercalated discs delineated
Collagen and bone – yellow to yellow green
Mucin – blue
Ground substance – blue green
Cytoplasm – pink to brownish to red

Materials

Stock solution A

Material	Amount
Orcein	1	g
Hydrochloric acid, conc.	1	mL
Ethanol, 70%	500	mL

Stock solution B

Material	Amount
Hematoxylin	8	g
Ethanol, absolute	160	mL

Stock solution C

Material	Amount
Ferric chloride	9.6	g
Distilled water	90	mL

Stock solution D

Material	Amount
Iodine	1	g
Potassium iodide	2	g
Distilled water	97	mL

Working elastic solution

Material	Amount
Stock solution A	25	mL
Stock solution B	8	mL
Stock solution C	5	mL
Stock solution D	5	mL

Differentiator

Material	Amount
Stock C	10	mL
Distilled water	40	mL

Alcian blue

Material	Amount
Alcian blue	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Ammoniated ethanol

Material	Amount
Strong ammonia	5	mL
Ethanol, 95%	95	mL

Plasma stain

Material	Amount
Woodstain scarlet 1% aqu.	4	mL
Acid fuchsin 1% aqu.	1	mL
Acetic acid, 0.5%	95	mL

Polyacid

Material	Amount
Phosphotungstic acid	5	g
Distilled water	100	mL

Fiber stain

Material	Amount
Spanish saffron	6	g
Ethanol, absolute	96	mL

Preparation

Incubate together in a sealed container at 56°C for 48 hours. Cool.

Tissue Sample

Paraffin sections at 4-6µ are suitable. Bouin’s fixation is preferred. 10% neutral buffered formalin is satisfactory. If formalin is used, refix sections with Bouin’s fluid for one hour at 56°C, then wash in running tap water to remove the yellow colour.

Protocol

Bring sections to water via xylene and ethanol.
Stain in alcian blue for 20 min.
Rinse in distilled water.
Place in ammoniated ethanol for 10 min at 56°C.
Wash in running tap water for 2 min.
Rinse in distilled water.
Stain in working elastic solution for 2 hours.
Wash in running water until collagen is clear and elastic prominent.
Rinse in distilled water.
Place in the plasma stain for 3 minutes.
Place in 0.5% aqueous acetic acid for 30 seconds.
Differentiate in the polyacid until collagen is clear and ground substance is blue.
Rinse in 0.5% aqueous acetic acid for 30 seconds.
Place in three changes of absolute ethanol for 1 minute each.
Place in the fiber stain for 8 minutes.
Dehydrate quickly in absolute ethanol, 2 changes.
Clear in xylene, three changes.
Coverslip with a resinous mounting medium.

Notes

The working elastic solution should be used only once, then discarded.
If the elastic fibers are not clearly delineated at step 8 and the background is not clean, place in the differentiator for a few minutes, then wash well in tap water until they are sharp.
Differentiation with the polyacid at step 12 takes from 3-10 minutes.
The fiber stain (alcoholic saffron) may be used repeatedly until staining intensity decreases. It is important that this solution not be contaminated with water. Place some Drierite into it to keep it anhydrous.

Safety Note

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

References

Silverman, J., (1972)
Histologic v 2, N° 2.

March 30, 2023

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Hollande’s Trichrome for Mitoses, Keratin and Collagen

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

Protocol

Hollande's Trichrome

for Mitoses, Keratin and Collagen

steps

materials

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Materials

An alum hematoxylin
Solution A
Material Amount
Basic fuchsin 1 g
Ethanol, 70% 100 mL
Solution B
Material Amount
Hydrochloric acid, conc. 0.1 mL
Ethanol, 70% 100 mL
Solution C
Material Amount
Phosphomolybdic acid 1 g
Distilled water 100 mL
Solution D
Material Amount
Orange G to saturation
Distilled water 100 mL
Solution E
Material Amount
Light green SF yellowish 0.2 g
Distilled water 100 mL

Material	Amount
Basic fuchsin	1	g
Ethanol, 70%	100	mL

Material	Amount
Hydrochloric acid, conc.	0.1	mL
Ethanol, 70%	100	mL

Material	Amount
Phosphomolybdic acid	1	g
Distilled water	100	mL

Material	Amount
Orange G	to	saturation
Distilled water	100	mL

Material	Amount
Light green SF yellowish	0.2	g
Distilled water	100	mL

Tissue Sample

Fixation requirements are unspecified, but likely many fixatives are satisfactory. 5µ paraffin sections of neutral buffered formalin fixed tissue are likely suitable. Many trichrome stains benefit from Bouin or mercuric chloride fixation of tissue or sections.

Protocol

Bring sections to water via xylene and ethanol.
Stain nuclei with an alum hematoxylin nuclear stain and blue.
Place into solution A for 6-12 hours.
Wash with water for 5 minutes.
Place into solution B, a few seconds, until clouds of dye stop being extracted.
Wash well with water.
Place into solution C for 5 minutes.
Rinse with water.
Place into solution D for 5 minutes.
Place into fresh solution E, 30-60 seconds, until differentiated.
Dehydrate with amyl alcohol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Mitoses – red
Cartilage – purple
Erythrocytes – orange
Keratin – orange
Collagen – green

Notes

Amyl alcohol was recommended for dehydration. If this is not available rapid dehydration with ethanol may suffice. Often t-butanol dehydrates satisfactorily without extracting excess dye.
The method specified clearing with benzene. For safety reasons this should be avoided. Xylene or toluene should be quite satisfactory.
Magenta was specified for solution A. This is an obsolete synonym for basic fuchsin.

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:
Hollande, (1912)
Archives de zoologie expérimentale et générale, v.10, pp.62

March 30, 2023

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Gomori’s Trichrome for Muscle and Collagen

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

Protocol

Gomori's Trichrome

for Muscle and Collagen

steps

materials

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Materials

Weigert’s iron hematoxylin or equivalent
Solution A
Material Amount
Chromotrope 2R 0.6 g
Fast green FCF 0.3 g
Phosphotungstic acid 0.6 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Solution B
Material Amount
Acetic acid, glacial 0.2 mL
Distilled water 100 mL

Material	Amount
Chromotrope 2R	0.6	g
Fast green FCF	0.3	g
Phosphotungstic acid	0.6	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Material	Amount
Acetic acid, glacial	0.2	mL
Distilled water	100	mL

Tissue Sample

Protocol

Bring sections to water via xylene and ethanol.
Stain nuclei with an acid resistant nuclear stain.
Place into solution A for 5-20 minute.
Quickly rinse off stain with distilled water.
Rinse with solution B.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – black
Cytoplasm – red
Collagen – green

Notes

The time in solution A should be determined empirically. Once a satisfactory stain has been achieved, the time should remain constant. Any change in fixation, processing or section thickness warrants reviewing the staining time.
Light green SF yellowish can replace Fast green FCF.
Wheatley recommended this trichrome for protozoa in intestinal smears.
Engel & Cunningham modified this method to differentiate muscle fibre types in cryostat sections.

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:
Gomori, (1950),
Technical Bulletin, vol.20, pp.77
And:
Wheatley, (1951)
Technical Bulletin, vol.21, pp.92
Humason, G. L., (1967)
Animal tissue techniques, Ed. 2
W. H. Freeman and Company, San Francisco, Ca, USA

March 30, 2023

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Heidenhain’s Azan Trichrome for Muscle and Collagen

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

Protocol

Heidenhain's Azan Trichrome

for Muscle and Collagen

steps

materials

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Materials

Solution A

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

Solution B

Material	Amount
Aniline	0.1	mL
Ethanol, 95%	100	mL

Solution C

Material	Amount
Hydrochloric acid	0.1	mL
Ethanol, 100%	100	mL

Solution D

Material	Amount
Phosphomolybdic acid	5	g
Distilled water	100	mL

Solution E

Material	Amount
Orange G	2	g
Aniline blue	0.5	g
Acetic acid, glacial	7.5	mL
Distilled water	100	mL

Tissue Sample

Protocol

Bring sections to water via xylene and ethanol.
Place into solution A at 50°C for 1 hour.
Rinse quickly with distilled water.
Place into solution B to differentiate nuclei. Dip into solution C before examining.
Rinse well with distilled water.
Place into solution D for 2 hours.
Place into solution E for 2-3 hours.
Rinse with distilled water.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – Red
Muscle – Orange
Collagen – 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:
Heidenhain, (1905)
Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik,
v.22, pp.339

March 30, 2023

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Kricheski’s Trichrome for Muscle and Collagen

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

Protocol

Kricheski's Trichrome

for Muscle and Collagen

steps

materials

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Materials

Solution A

Material	Amount
Acid fuchsin	0.25	g
Distilled water	100	mL

Solution B

Material	Amount
Methyl blue, 1% aqueous	30	mL
Orange G, 1% aqueous	30	mL
Phosphomolybdic acid, 1% aqueous	30	mL

Tissue Sample

Protocol

Bring sections to water via xylene and ethanol.
Place into solution A for 1-3 minute.
Rinse well with distilled water.
Place into solution B for 3-5 minutes.
Dip 2 or 3 times in 70% ethanol.
Dehydrate and differentiate with ethanol for 1-3 minutes.
Clear with xylene and mount with a resinous medium.
Bring sections to water via xylene and ethanol.

Expected Results

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

Notes

Although this method does not specify that an acid resistant nuclear stain be used, Weigert’s iron hematoxylin or equivalent could be inserted before step 2, lightly differentiated and blued. The nuclei would then be black.

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:
Kricheski, (1931)
Stain technology, v.6, pp.97

March 30, 2023

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Ladewig’s Trichrome for Muscle and Collagen

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

Protocol

Ladewig's Trichrome

for Muscle and Collagen

steps

materials

print

Materials

Weigert’s iron hematoxylin or equivalent
Solution A
Material Amount
Phosphotungstic acid 1 g
Distilled water 100 mL
Solution B
Material Amount
Methyl blue 0.5 g
Orange G 2 g
Acid fuchsin 1 g
Oxalic acid 0.6 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. Many trichrome stains benefit from Bouin or mercuric chloride fixation. Sections of formalin fixed tiddue may benefit from secondary fixation in Bouin’s fluid.

Protocol

Bring sections to water via xylene and ethanol.
Stain nuclei with Weigert’s iron hematoxylin for 3-5 minutes.
Rinse with water.
Place into solution A for 2 minute.
Rinse with distilled water.
Place into solution B for 4 minutes.
Rinse quickly with distilled water.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

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

Notes

Times may need to be adjusted, particularly if fixatives other than formalin have been used.

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:
Ladewig, (1938)
Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik,
v.55, pp.215

March 30, 2023

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Material	Amount
Methyl blue	0.5	g
Orange G	2	g
Acid fuchsin	1	g
Oxalic acid	0.6	g
Distilled water	100	mL