Slidders' Orange-Fuchsin-Green (OFG)

For Pituitary Cells

steps

materials

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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

steps

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

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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Maxwell’s Trichrome for Pituitary Cells

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

Protocol

Maxwell's Trichrome

for Pituitary Cells

steps

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

steps

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
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

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

steps

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

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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Lewis and Miller’s Trichrome for Pituitary cells

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

Protocol

Lewis and Miller's Trichrome

for Pituitary cells

steps

materials

print

This is a modification of Kricheski’s method.

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

5µ paraffin sections of neutral buffered formalin fixed tissue are likely suitable. Other fixatives are also 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 30 minute.
Rinse well with distilled water.
Place into solution B for 24 hours.
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.

Expected Results

Nuclei – red
Acidophils – orange red
Basophils – blue
Chromophobes – Pale grey

Notes

Although not specified, an acid resistant nuclear stain such as Weigert’s iron hematoxylin could be inserted prior to staining with solution A.

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
And:
Lewis and Miller, (1938)
Stain technology, v.14, pp.111

March 30, 2023

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Lendrum & McFarlane’s Trichrome for Muscle and Collagen

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

Protocol

Lendrum & McFarlane's Trichrome

for Muscle and Collagen

steps

materials

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Materials

Celestine blue-hemalum
Solution A
Material Amount
Picric acid 1 g
Orange G 0.2 g
Ethanol, 95% 80 mL
Water 20 mL
Solution B
Material Amount
Acid fuchsin 0.5 g
Ponceau 2R 0.5 g
Sodium sulphate 0.25 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Solution C
Material Amount
Acetic acid, glacial 1 mL
Distilled water 99 mL
Solution D
Material Amount
Phosphomolybdic acid 1 g
Distilled water 100 mL
Solution E
Material Amount for Variation I Amount for Variation II
Aniline blue 2 g –
Fast green FCF – 2 g
Acetic acid, glacial 1 mL 1 mL
Distilled water 100 mL 100 mL

Material	Amount
Picric acid	1	g
Orange G	0.2	g
Ethanol, 95%	80	mL
Water	20	mL

Material	Amount
Acid fuchsin	0.5	g
Ponceau 2R	0.5	g
Sodium sulphate	0.25	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Material	Amount
Phosphomolybdic acid	1	g
Distilled water	100	mL

Material	Amount for Variation I	Amount for Variation II
Aniline blue	2	g	–
Fast green FCF	–		2	g
Acetic acid, glacial	1	mL	1	mL
Distilled water	100	mL	100	mL

Tissue Sample

Most trichrome stains benefit from picric acid or mercuric chloride fixation. 5µ paraffin sections of neutral buffered formalin fixed tissue would probably be suitable, although secondary fixation of sections in Bouin’s fluid overnight or at 56°C for an hour would undoubtedly enhance staining.

Protocol

Bring sections to water via xylene and ethanol.
Stain nuclei with the celestine blue-hemalum sequence.
Rinse well with water.
Place into solution A for 2 minutes to overnight.
Rinse with water.
Place into solution B for 1-5 minutes.
Rinse with solution C.
Place into solution D until collagen is almost decolorized.
Place into solution E for 2-10 minutes.
Rinse with solution C.
Return to solution E for 3-5 minutes.
Dehydrate with absolute ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

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

Notes

The method appears to call for solution A to be applied for 2 minutes to overnight, although the instructions in Gray are not completely clear. It should be noted that this solution contains sufficient picric acid to act as a fixative as well as to stain erythrocytes, and overnight treatment might function partly to bring about some secondary fixation. For tissue well fixed with Bouin or a mercuric chloride fixative, 2 minutes should be adequate.
Solution A calls for a weight of 1 gram of (dry) picric acid. An equivalent solution using a saturated ethanolic solution would be:
Material Amount
Picric acid, sat. ethanolic 12 mL
Orange G 0.2 g
Ethanol, 95% 68 mL
Water 20 mL

Material	Amount
Picric acid, sat. ethanolic	12	mL
Orange G	0.2	g
Ethanol, 95%	68	mL
Water	20	mL

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:
Lendrum and McFarlane, (1940)
Journal of Pathology and Bacteriology, v. 50, pp. 381

March 30, 2023

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Koneff’s Trichrome for Pituitary Cells

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

Protocol

Koneff's Trichrome

for Pituitary Cells

steps

materials

print

Materials

Solution A

Material	Amount
Aniline	0.1	mL
Ethanol, 90%	100	mL

Solution B

Material	Amount
Acetic acid, glacial	1	mL
Ethanol, 90%	100	mL

Solution C

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

Solution D

Material	Amount
Aniline	0.06	mL
Ethanol, 90%	100	mL

Solution E

Material	Amount
Phosphotungstic acid	5	g
Distilled water	100	mL

Solution F

Material	Amount
Aniline blue	0.5	g
Orange G	2	g
Oxalic acid	2	g
Phosphotungstic acid	0.05	g
Distilled water	100	mL

Solution G

Material	Amount
Acetic acid	1	mL
Distilled water	100	mL

Tissue Sample

The instructions specify fixation in see Baley’s fixative. Other mercuric-chrome fixatives would probably be satisfactory. The instructions also specify 3-4 µ celloidin sections which have been attached to a slide, and from which the celloidin has been removed. Possibly, similar paraffin sections would be satisfactory.

Baley’s fixative consists of:

Material	Amount
Mercuric chloride	3.4	g
Potassium dichromate	3.4	g
Concentrated formalin (40%)	25	mL
Water	225	mL
Return to fixation instructions.

Protocol

Attach sections to slides and remove celloidin, or dewax sections with xylene and bring to ethanol.
Although not specified, a water rinse followed by the iodine thiosulphate sequence and a second water wash, to remove mercury pigment would be advantageous at this point.
Wash with 70% ethanol.
Place into solution A for 45 minutes.
Place into solution B for 1-2 minutes.
Place into solution C for 2 hours at 56°C.
Wash with water.
Place into solution D until nuclei are red and the cytoplasm is pink.
Place into solution B for 1-2 minutes.
Place into solution E for 4 hours
Place into solution F for 4 hours until basophils are blue.
Return to solution E for 3-5 minutes.
Wash with water.
Rinse with solution G.
Wash with water.
Dehydrate with absolute ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – red
Acidophils – orange-red
Basophils – blue
Chromophobes – pale grey

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:
Koneff, (1938)
Stain Technology, v.13, pp.49

March 30, 2023

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Klatskin’s Modification of Masson’s Trichrome for Liver

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

Protocol

Klatskin's Modification of Masson's Trichrome

for Liver

steps

materials

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Materials

Harris’ alum hematoxylin.
Solution A
Material Amount
Picric acid to saturation
Ethanol, 95% 100 mL
Solution B
Material Amount
Xylidine ponceau 1 g
Distilled water 99 mL
Glacial acetic acid 1 mL
Solution C
Material Amount
Phosphomolybdic acid 1 g
Distilled water 100 mL
Solution D
Material Amount
Aniline blue to saturation
Glacial acetic acid 2.5 mL
Distilled water 97.5 mL
Solution E
Material Amount
Acetic acid, glacial 1 mL
Distilled water 99 mL

Material	Amount
Picric acid	to saturation
Ethanol, 95%	100	mL

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

Material	Amount
Phosphomolybdic acid	1	g
Distilled water	100	mL

Material	Amount
Aniline blue	to saturation
Glacial acetic acid	2.5	mL
Distilled water	97.5	mL

Tissue Sample

3-5 micron paraffin sections of formalin fixed liver.

Protocol

Bring sections to water via xylene and ethanol
Stain with Harris’ hemalum for 10 minutes.
Rinse with 95% ethanol.
Place in solution A for 10 minutes.
Wash with water for 10 minutes.
Place in solution B for 5 minutes.
Place in solution C for 5 minutes.
Place in solution D for 2 minutes.
Replace in solution C for 5 minutes.
Place in solution E for 5 minutes.
Place in 70% ethanol for 2 minutes.
Dehydrate with ethanol, clear and mount with a resinous medium.

Expected Results

Cytoplasm – red
Erythrocytes – red
Muscle – red
Collagen – blue
Nuclei – brown

Notes

Aniline blue, is a mixture of two dyes, methyl blue, and water blue. Either may usually be substituted satisfactorily.
Although recommended for liver sections, this modification may also be used as a general replaceement for Masson’s method.

Safety Note

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

References

Petersen, K.F., West, A.B., Reuben, A., Rothman, D. and Shulman, G.I. (1996)
Noninvasive Assessment of Hepatic Triglyceride Content in Humans With 13C Nuclear Magnetic Resonance Spectroscopy.
Hepatology, V. 24, p 114-117

March 30, 2023

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Haythorne’s Trichrome for Muscle and Collagen

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

Protocol

Haythorne's Trichrome

for Muscle and Collagen

steps

materials

print

Materials

Böhmer’s alum hematoxylin
Solution A
Material Amount
Orange G 0.8 g
Ferric ammonium sulphate 5 g
Hydrochloric acid 0.06 mL
Ethanol, 95% 4 mL
Distilled water 100 mL
Dissolve the iron alum into 25 mL of the water.

Combine the remaining ingredients.

Mix the two solutions together and filter.
Solution B
Material Amount
Acid fuchsin 1 g
Distilled water 100 mL
Solution C
Material Amount
Aniline blue 2.5 g
Orange G 2.5 g
Phosphomolybdic acid to saturation
Distilled water 100 mL

Tissue Sample

Zenker fixation was recommended, and 5µ paraffin sections would be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixation could probably be used with secondary fixation of sections in Bouin’s fluid.

Protocol

Bring sections to water via xylene and ethanol.
Stain nuclei with Böhmer’s hematoxylin for 30 minutes.
Place into solution A for 2 minutes.
Wash with water for 5 minutes.
Place into solution B for 3 minutes.
Blot.
Place into solution C for 20 minutes.
Blot.
Rinse quickly with 95% ethanol.
Dehydrate and differentiate with absolute ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – reddish black
Erythrocytes – orange
Keratin – orange
Cytoplasm – red
Collagen – blue
Cartilage – blue

Notes

The iron alum in solution A probably serves to mordant the Böhmer’s hematoxylin, converting it into an iron hematoxylin, thus making the nuclear stain acid resistant.
Böhmer’s stain is an obsolete alum hematoxylin and a more modern progressive hemalum, such as Mayer’s, may be suitable.
Alternatively, Böhmer’s hematoxylin could probably be replaced with an acid resistant nuclear stain such as the celestine blue hemalum sequence, or Weigert’s iron hematoxylin. If so, the iron alum in solution A would likely be redundant (and the method would no longer be Haythorne’s).

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:
Haythorne, (1916)
Bulletin of the International Association of Medical Museums
and Journal of Technical Methods., v. 6, p. 61
Montreal, Que. Canada & Washington, DC, USA

March 30, 2023

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Material	Amount
Orange G	0.8	g
Ferric ammonium sulphate	5	g
Hydrochloric acid	0.06	mL
Ethanol, 95%	4	mL
Distilled water	100	mL