Slidders' Orange-Fuchsin-Green (OFG)

For Pituitary Cells

14

steps

10

materials

print

Materials

Lendrum’s celestine blue
A progressive hemalum, such as Mayer’s
Solution A
Material Amount
Orange G 0.05 g
Ethanol, 95% 100 mL
Phosphotungstic acid 0.5 g
Solution B
Material Amount
Acid fuchsin 0.5 g
Acetic acid, glacial 0.5 mL
Distilled water 100 mL
Solution C
Material Amount
Phosphotungstic acid 1 g
Distilled water 100 mL
Solution D
Material Amount
Light green SF yellowish 1.5 g
Acetic acid, glacial 1.5 mL
Distilled water 100 mL

Material	Amount
Orange G	0.05	g
Ethanol, 95%	100	mL
Phosphotungstic acid	0.5	g

Material	Amount
Acid fuchsin	0.5	g
Acetic acid, glacial	0.5	mL
Distilled water	100	mL

Material	Amount
Phosphotungstic acid	1	g
Distilled water	100	mL

Material	Amount
Light green SF yellowish	1.5	g
Acetic acid, glacial	1.5	mL
Distilled water	100	mL

Tissue Sample

Although 5µ paraffin sections of neutral buffered formalin fixed tissue are likely adequate, superior results are obtained with fixation in formal sublimate, preferably about 48 hours. Other fixatives may be satisfactory. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

Protocol

Bring sections to water via xylene and ethanol.
Remove mercury pigment.
Stain nuclei with Lendrum’s celestine blue-hemalum.
Rinse with 95% ethanol.
Place into solution A for 2 minutes.
Rinse well with distilled water
Place into solution B for 2-5 minutes, until basophiles are red.
Rinse with distilled water.
Differentiate with solution C for 5 minutes.
Rinse with distilled water.
Place into solution D for 2 minutes
Rinse well with distilled water.
Dehydrate with ethanols.
Clear with xylene and mount with a resinous medium.

Expected Results

Best results are obtained if staining times are controlled visually.

Notes

Nuclei – black
Erythrocytes – orange
Collagen – green
Acidophils – orange
Basophils – red
Chromophobes – pale gray-green

Safety Note

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

References

Putt, F. A.
Manual of Histopathological Staining Methods
John Wiley & Sons, New York, NY., USA
Citing:
Slidders, W. J., (1961)
Journal of Pathology and Bacteriology, v. 82, p. 532
London, England.

March 30, 2023

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Weiss’ Trichrome for Muscle and Collagen

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

Protocol

Weiss' Trichrome

for Muscle and Collagen

8

steps

7

materials

print

Materials

Weigert’s iron hematoxylin or equivalent
Solution A
Material Amount
Acid fuchsin 0.04 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

Material	Amount
Acid fuchsin	0.04	g
Distilled water	100	mL

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. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

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 4 minutes.
Wash briefly with distilled water.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – black
Erythrocytes – yellow
Cytoplasm – red
Collagen – Blue

Notes

The original recommended nuclear staining in Delafield’s alum hematoxylin, with blueing.
This differs from Brillmeyer’s trichrome by using a weaker acid fuchsin solution and staining in aniline blue for a much shorter time.

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:
Weiss, (1932)
Stain Technology, v. 7, pp. 131

March 30, 2023

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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

9

steps

8

materials

print

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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Wallart & Honette’s Trichrome for Connective Tissues

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

Protocol

Wallart & Honette's Trichrome

for Connective Tissues

9

steps

6

materials

print

Materials

Stock solution 1

Material	Amount
Acid fuchsin	1	g
Acetic acid	1	mL
Distilled water	100	mL

Stock solution 2

Material	Amount
Fast yellow	3	g
Acetic acid	1	mL
Distilled water	100	mL

Stock solution 3

Material	Amount
Phosphomolybdic acid	1	g
Distilled water	100	mL

Working solution A

Material	Amount
Stock solution 1	30	mL
Stock solution 2	30	mL
Stock solution 3	30	mL

Working solution B

Material	Amount
Acetic acid	1	mL
Distilled water	100	mL

Working solution C

Material	Amount
Acetic acid	1	mL
Ethanol 100%	100	mL

Tissue Sample

5µ paraffin sections of formalin fixed tissue are suitable. Many 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 acid resistant nuclear stain.
Rinse well with tap water.
Place into solution A for 5 minute.
Rinse quickly with distilled water.
Place into solution B for 5 minutes.
With a pipette, drop solution C onto the slide for 30 seconds.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Elastic – pink
Cytoplasm – red
Collagen – yellow
Nuclei – 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:
Wallart and Honette, (1934)
Bulletin d’histologie appliquée à la physiologie et à la pathologie et de technique microscopique, v. 10, p. 404.

March 30, 2023

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Pollak’s Trichrome for Muscle and Collagen

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

Protocol

Pollak's Trichrome

for Muscle and Collagen

6

steps

11

materials

print

Materials

Weigert’s iron hematoxylin or equivalent
Solution A
Material Amount
Orange G 0.25 g
Ponceau 2R 0.33 g
Acid fuchsin 0.17 g
Light green SF 0.15 g
Phosphotungstic acid 0.5 g
Phosphomolybdic acid 0.5 g
Acetic acid, glacial 1 mL
Ethanol, 95% 50 mL
Distilled water 50 mL
Solution B
Material Amount
Acetic acid, glacial 0.2 mL
Distilled water 100 mL

Material	Amount
Orange G	0.25	g
Ponceau 2R	0.33	g
Acid fuchsin	0.17	g
Light green SF	0.15	g
Phosphotungstic acid	0.5	g
Phosphomolybdic acid	0.5	g
Acetic acid, glacial	1	mL
Ethanol, 95%	50	mL
Distilled water	50	mL

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

Preparation of Solution A

Mix the water, ethanol and acetic acid together.
Divide into four, and dissolve as follows:
1. Phosphomolybdic acid
2. Phosphotungstic acid and orange G
3. Light green SF
4. Ponceau 2R and acid fuchsin
Combine all solutions and filter.

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 Weigert’s iron hematoxylin or equivalent.
Place into solution A for 3-7 minute.
Place into solution B until differentiated.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – black
Erythrocytes – yellow
Cytoplasm – red
Muscle – red
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:
Pollack, (1944)
Archives of pathology and laboratory medicine, v. 37, pp. 294
Chicago, USA

March 30, 2023

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Sweat, Meloan & Puchtler’s One Step Trichrome

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

Protocol

Sweat, Meloan & Puchtler's

One Step Trichrome

7

steps

6

materials

print

Materials

Solution A

Material	Amount
Chromotrope 2R	0.6	g
Aniline blue	0.6	g
Phosphomolybdic acid	1	g
Hydrochloric acid, conc.	1	mL
Distilled water	100	mL

Solution B

Material	Amount
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 in Bouin’s fixative at 56°C for one hour.
Wash with running tap water 3 to 4 minutes to remove yellow.
Place into solution A for 1 minute.
Rinse with solution B for 30 seconds.
Dehydrate rapidly with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei, cytoplasm, muscle, fibrin & elastic – red
Collagen – blue

Notes

A nuclear stain is not used as the hydrochloric acid will remove it.

Safety Note

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

References

Culling, C.F.A., Alison, R.T. and Barr, W.T. (1985)
Cellular Pathology Technique, 4th ed.
Butterworths, London, UK.

March 30, 2023

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Maxwell’s Trichrome for Pituitary Cells

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

Protocol

Maxwell's Trichrome

for Pituitary Cells

12

steps

12

materials

print

Materials

Solution A

Material	Amount
Acid fuchsin	1	g
Distilled water	100	mL

Solution B

Material	Amount
Ammonia (0.880)	0.02	mL
Distilled water	100	mL

Solution C

Material	Amount
Hydrochloric acid (Conc.)	0.1	mL
Distilled water	100	mL

Solution D

Material	Amount
Phosphomolybdic acid	0.5	g
Distilled water	100	mL

Solution E

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

Maxwell’s fluid

Material	Amount
Cedarwood oil	30	mL
Thyme oil	40	mL
Xylene	15	mL
Ethanol, absolute	15	mL

Tissue Sample

Paraffin sections at 5µ are suitable. Heidenhain’s (Zenker formol) fixation was specified. Other mercuric chloride fixatives would likely be satisfactory.

Protocol

Bring sections to water via xylene and ethanol.
Place into solution A for 30 minute.
Rinse with distilled water.
Place into solution B until differentiated.
Place into solution C for a few seconds.
Place into solution D for 3 minutes.
Place into solution E for 60 minutes.
Rinse with distilled water.
Differentiate with 95% ethanol.
Dehydrate with ethanol.
Clear with Maxwell’s fluid or xylene.
Mount with Canada balsam or other resinous medium.

Expected Results

Nuclei – red
Acidophils – orange red
basophils – blue

Notes

Maxwell’s fluid was originally specified for clearing, but this is probably not necessary.

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:
Maxwell, (1938)
Stain Technology, vol. 13, pp. 93

March 30, 2023

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McFarlane’s Trichrome 3rd Variant for Muscle and Collagen

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

Protocol

McFarlane's Trichrome 3rd Variant

for Muscle and Collagen

14

steps

9

materials

print

Materials

Regaud’s iron hematoxylin or equivalent
Solution A
Material Amount
Picric acid 1 g
Orange G 0.25 g
Ethanol, 95% 80 mL
Distilled water 20 mL
Solution B
Material Amount
Acid fuchsin 0.25 g
Ponceau 2R 0.25 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Solution C
Material Amount
Acetic acid, glacial 2 mL
Distilled water 98 mL
Solution D
Material Amount
Picric acid 1 g
Phosphotungstic acid 10 g
Ethanol, 95% 80 mL
Distilled water 20 mL
Solution E
Material Amount
Aniline blue 2.5 g
Acetic acid, glacial 2.5 mL
Distilled water 97.5 mL
Solution F
Material Amount
Picric acid 0.5 g
Phosphotungstic acid 5 g
Ethanol, 95% 20 mL
Distilled water 801 mL

Material	Amount
Picric acid	1	g
Orange G	0.25	g
Ethanol, 95%	80	mL
Distilled water	20	mL

Material	Amount
Acid fuchsin	0.25	g
Ponceau 2R	0.25	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Material	Amount
Acetic acid, glacial	2	mL
Distilled water	98	mL

Material	Amount
Picric acid	1	g
Phosphotungstic acid	10	g
Ethanol, 95%	80	mL
Distilled water	20	mL

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

Material	Amount
Picric acid	0.5	g
Phosphotungstic acid	5	g
Ethanol, 95%	20	mL
Distilled water	801	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 Régaud’s iron hematoxylin, but do not differentiate with iron alum.
Place into solution A until nuclei are differentiated.
Wash with water until erythrocytes alone remain yellow.
Place into solution B for 5-10 minutes.
Rinse with solution C.
Place into solution D for 5 minutes until differentiated.
Rinse with solution C.
Place into solution E for 10 minutes.
Rinse with solution C.
Place into solution F until differentiated.
Wash with solution C.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

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

Notes

McFarlane also has another multi-step method and a one step method.
Solutions A, D and F require dry weights of picric acid. These could be provided from a saturated ethanolic (1 g in 12 mL) solution. Alternate formulas are:
Solution A
Material Amount
Picric acid, sat. alc. 12 mL
Ethanol, 95% 68 mL
Distilled water 20 mL
Orange G 0.25 g
Solution D
Material Amount
Picric acid, sat. alc. 12 mL
Ethanol, 95% 68 mL
Distilled water 20 mL
Phosphotungstic acid 10 g
Solution F
Material Amount
Picric acid, sat. alc. 6 mL
Ethanol, 95% 14 mL
Distilled water 80 mL
Phosphotungstic acid 5 g

Material	Amount
Picric acid, sat. alc.	12	mL
Ethanol, 95%	68	mL
Distilled water	20	mL
Orange G	0.25	g

Material	Amount
Picric acid, sat. alc.	12	mL
Ethanol, 95%	68	mL
Distilled water	20	mL
Phosphotungstic acid	10	g

Material	Amount
Picric acid, sat. alc.	6	mL
Ethanol, 95%	14	mL
Distilled water	80	mL
Phosphotungstic acid	5	g

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:
McFarlane, (1944)
Stain Technology, v. 19, pp. 29

March 30, 2023

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McFarlane’s Trichrome 2nd Variant for Muscle and Collagen

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

Protocol

McFarlane's Trichrome 2nd Variant

for Muscle and Collagen

12

steps

9

materials

print

Materials

Weigert’s iron hematoxylin or equivalent
Solution A
Material Amount
Picric acid 0.2 g
Acid fuchsin 0.8 g
Acetic acid, glacial 2 mL
Distilled water 98 mL
Solution B
Material Amount
Acetic acid, glacial 2 mL
Distilled water 98 mL
Solution C
Material Amount
Picric acid 1 g
Phosphotungstic acid 10 g
Ethanol, 95% 40 mL
Distilled water 60 mL
Solution D
Material Amount
Aniline blue 2.5 mL
Acetic acid, glacial 2.5 g
Distilled water 97.5 mL
Solution E
Material Amount
Picric acid 0.25 g
Phosphotungstic acid 2.5 g
Ethanol, 95% 10 mL
Distilled water 90 mL

Material	Amount
Picric acid	0.2	g
Acid fuchsin	0.8	g
Acetic acid, glacial	2	mL
Distilled water	98	mL

Material	Amount
Acetic acid, glacial	2	mL
Distilled water	98	mL

Material	Amount
Picric acid	1	g
Phosphotungstic acid	10	g
Ethanol, 95%	40	mL
Distilled water	60	mL

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

Material	Amount
Picric acid	0.25	g
Phosphotungstic acid	2.5	g
Ethanol, 95%	10	mL
Distilled water	90	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 Weigert’s iron hematoxylin or equivalent.
Place into solution A for 5 minute.
Rinse with solution B.
Place into solution C for 5 minutes.
Rinse with distilled water.
Place into solution D for 5-10 minutes.
Rinse with solution B.
Place into solution E for 5 minutes.
Wash with solution B.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

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

Notes

McFarlane also has another multi-step method and a one step method.
Solutions A, C and E require dry weights of picric acid. These could be provided from saturated aqueous (1 g in 78 mL) or ethanolic (1 g in 12 mL) solutions. Alternate formulas are:
Solution A
Material Amount
Picric acid, sat. alc. 16 mL
Distilled water 82 mL
Acetic acid, glacial 2 mL
Acid fuchsin 0.8 g
Solution C
Material Amount
Picric acid, sat. alc. 12 mL
Distilled water 60 mL
Ethanol, 95% 28 mL
Phosphotungstic acid 10 g
Solution E
Material Amount
Picric acid, sat. alc. 20 mL
Distilled water 70 mL
Ethanol, 95% 10 mL
Phosphotungstic acid 2.5 g

Material	Amount
Picric acid, sat. alc.	16	mL
Distilled water	82	mL
Acetic acid, glacial	2	mL
Acid fuchsin	0.8	g

Material	Amount
Picric acid, sat. alc.	12	mL
Distilled water	60	mL
Ethanol, 95%	28	mL
Phosphotungstic acid	10	g

Material	Amount
Picric acid, sat. alc.	20	mL
Distilled water	70	mL
Ethanol, 95%	10	mL
Phosphotungstic acid	2.5	g

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:
McFarlane, (1944)
Stain Technology, v. 19, pp. 29

March 30, 2023

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McFarlane’s Trichrome 1st Variant for Muscle and Collagen

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

Protocol

McFarlane's Trichrome 1st Variant

for Muscle and Collagen

10

steps

9

materials

print

Materials

Weigert’s iron hematoxylin or equivalent
Solution A
Material Amount
Picric acid 0.2 g
Acid fuchsin 1 g
Aniline blue 2 g
Phosphotungstic acid 1 g
Acetic acid, glacial 2 mL
Distilled water 98 mL
Solution B
Material Amount
Acetic acid, glacial 2 mL
Distilled water 100 mL
Solution C
Material Amount
Picric acid 0.25 g
Phosphotungstic acid 2.5 g
Ethanol, 95% 10 mL
Distilled water 90 mL

Material	Amount
Picric acid	0.2	g
Acid fuchsin	1	g
Aniline blue	2	g
Phosphotungstic acid	1	g
Acetic acid, glacial	2	mL
Distilled water	98	mL

Material	Amount
Acetic acid, glacial	2	mL
Distilled water	100	mL

Material	Amount
Picric acid	0.25	g
Phosphotungstic acid	2.5	g
Ethanol, 95%	10	mL
Distilled water	90	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 Weigert’s iron hematoxylin or equivalent.
Place into solution A for 5 minute.
Rinse with solution B.
Rinse with distilled water.
Place into solution C until differentiated.
Rinse with distilled water.
Wash with solution B.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

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

Notes

McFarlane also published two other, multi-step, trichrome methods. One is simpler than the other.
Solutions A and C require dry weights of picric acid. This could be provided from a saturated aqueous solution (1 g in 78 mL). Alternate formulas are:
Solution A
Material Amount
Picric acid, sat. aqueous 16 mL
Distilled water 82 mL
Acetic acid, glacial 2 mL
Acid fuchsin 1 g
Aniline blue 2 g
Phosphotungstic acid 1 g
Solution C
Material Amount
Picric acid, sat. aqueous 20 mL
Distilled water 70 mL
Ethanol, 95% 10 mL
Phosphotungstic acid 2.5 g

Material	Amount
Picric acid, sat. aqueous	16	mL
Distilled water	82	mL
Acetic acid, glacial	2	mL
Acid fuchsin	1	g
Aniline blue	2	g
Phosphotungstic acid	1	g

Material	Amount
Picric acid, sat. aqueous	20	mL
Distilled water	70	mL
Ethanol, 95%	10	mL
Phosphotungstic acid	2.5	g

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:
McFarlane, (1944)
Stain Technology, v. 19, pp. 29

March 30, 2023

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