Walter’s Trichrome for Elastic and Collagen

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

Protocol

Walter's Trichrome

for Elastic and Collagen

steps

materials

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Materials

Solution A

Material	Amount
Ferric ammonium sulphate	2.5	g
Distilled water	100	mL

Solution B

Material	Amount
Phosphotungstic acid	2	g
Distilled water	100	mL

Solution C

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

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 24 hours.
Rinse quickly with distilled water.
Place into solution B for 10 minutes.
Rinse quickly with distilled water.
Place into solution C for 15-20 minutes.
Rinse quickly with 95% ethanol until clouds of dye stop being extracted.
Dehydrate with 100% ethanol for 1 minute.
Clear with xylene and mount with a resinous medium.

Expected Results

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

Notes

This is a modification of Pasini’s method.

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:
Walter, (1930)
Zeitschrift für wissenschaftliche Mikroskopie und für mikroskipische Technik,
v. 46, pp. 458

March 30, 2023

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Taenzer-Unna Orcein for Elastic Fibres

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

Protocol

Taenzer-Unna Orcein

for Elastic Fibres

steps

materials

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Materials

Orcein solution

Material	Amount
Orcein	1	g
Ethanol, 70%	100	mL
Hydrochloric acid, conc.	1	mL

Acid ethanol

Material	Amount
Ethanol, 70%	100	mL
Hydrochloric acid, conc.	1	mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Protocol

Bring sections to water via xylene and ethanol.
Rinse with 70% ethanol.
Place into orcein solution for an appropriate time.
Optionally, treat with acid ethanol until collagen is unstained (a few seconds).
Wash with water
Counterstain if wished.
Dehydrate with 95% and absolute ethanols.
Clear with xylene and mount with a resinous medium.

Expected Results

Elastic fibres – purplish brown
Other tisse – as counterstained

Notes

Gray specifies 0.8 grams orcein, most others specify 1 gram.
Gray specifies 50% ethanol, Culling 70% and Carleton 80%. Others have recommended concentrations ranging from 65% to 100% (Lillie).
Staining times specified include 6-12 hours (Gray), overnight (Culling) and 30 minutes to 2 hours (Carleton).
Elevating the temperature (37°C Carleton, 56°C Culling) decreases staining time.
Recommended counterstains are van Gieson, methylene blue, H&E.

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:
von Kahlden, C., and Laurent, O. (1896)
Technique microscopique.
Carré, Paris, France
Drury, R.A.B. and Wallington, E.A., (1980)
Carleton’s histological technique Ed. 5
Oxford University Press, Oxford, UK.
Culling C.F.A., (1974)
Handbook of histopathological and histochemical techniques Ed. 3
Butterworth, London, UK.
Mallory, F. B. & Wright, J.H., (1904)
Pathological technique, Ed.3
W. B. Saunders, Philadelphia, USA.
Lillie, R.D., (1954)
Histopathologic technique and practical histochemistry Ed.2
Blakiston, New York, USA.

March 30, 2023

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Unna’s Orcein-Aniline Blue for Elastic

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

Protocol

Unna's Orcein-Aniline Blue

for Elastic

steps

materials

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Materials

A red nuclear stain
Solution A
Material Amount
Acetic acid, glacial 1 mL
Distilled water 1 L
Solution B
Material Amount
Aniline blue 0.5 g
Orcein 0.5 g
Acetic acid, glacial 2.5 mL
Ethanol, 100% 25 mL
Distilled water 50 mL
Glycerol 10 mL

Material	Amount
Aniline blue	0.5	g
Orcein	0.5	g
Acetic acid, glacial	2.5	mL
Ethanol, 100%	25	mL
Distilled water	50	mL
Glycerol	10	mL

Preparation

Dissolve the aniline blue in the water with heat.
Cool and filter.
Dissolve the orcein in the ethanol.
Add the acetic acid and glycerol.
Combine the solutions.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Protocol

Bring sections to water via xylene and ethanol.
Stain the nuclei with a red nuclear stain.
Place into solution A for a few minutes.
Place into solution B for 1-10 hours.
Differentiate with solution A until elastic fibres are clear.
Dehydrate with 95% and absolute ethanols.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – red
Elastic – reddish brown
Bone – reddish brown
Background – blue

Notes

Gatenby & Beams specified water blue rather than aniline blue
Unna’s method has been more commonly applied in conjunction with other methods, such as Pasini’s trichrome, with solution B (Unna’s solution) being incorporated into other staining solutions.

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

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

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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, 2% in 50% ethanol	30	mL
Acid fuchsin, sat. aqu.	4	mL
Unna’s elastic stain	30	mL
Glycerol	15	mL

Tissue Sample

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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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

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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 – 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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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

steps

materials

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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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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

steps

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

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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