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

print

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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Lendrum’s Phloxine Tartrazine for Viral Inclusion Bodies

By Carmen WongIntracytoplasmic Granules, Paneth Cells, Protocols, Stain Target, Stain Type, Trichrome Staining, Yellowsolve Staining

Protocol

Lendrum's Phloxine Tartrazine

for Viral Inclusion Bodies

steps

materials

print

This method is also used for the demonstration of Paneth cell granules, and may be used as a substitute for the HPS if the differentiation in the tartrazine solution is shortened to retain pink cytoplasm and muscle.

Materials

Mayer’s hemalum
Solution A
Material Amount
Phloxine B 0.5 g
Calcium chloride 0.5 g
Distilled water 100 mL
Solution B
Material Amount
Tartrazine to saturation
2-Ethoxy ethanol (cellosolve) 100 mL

Material	Amount
Phloxine B	0.5	g
Calcium chloride	0.5	g
Distilled water	100	mL

Material	Amount
Tartrazine	to saturation
2-Ethoxy ethanol (cellosolve)	100	mL

Tissue Sample

5µ paraffin sections of formal sublimate fixed tissue is preferred. Formalin fixed tissue is suitable. Other fixatives are likely to be satisfactory.

Protocol

Bring sections to water via xylene and ethanol.
Stain nuclei to medium density with hemalum.
Wash in tap water for 5 minutes. Blueing takes place in the phloxine solution.
Place in solution A for 20 minutes.
Rinse in tap water, blot almost dry. Some technologists rinse with cellosolve instead of blotting. The object is to remove all traces of water, as it interferes with the ability of tartrazine to extract phloxine and counterstain.
Rinse with solution B to remove remaining water. Discard solution.
Place in solution B until inclusions are red and all other tissue is yellow. The time varies considerably. Control microscopically.
Rinse thoroughly but briefly with absolute ethanol.
Do not rinse with water at this stage as it rapidly removes the tartrazine. Some technologists use cellosolve instead of ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Acidophil virus inclusion bodies – red
Paneth cell granules – red
Background – yellow

Notes

The 2-ethoxy ethanol must not be replaced by any other solvent.
The 2-ethoxy ethanol must be, and must remain, completely anhydrous.

Safety Note

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

References

Bancroft, J.D. and Stevens A. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.

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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Kostowiecki’s Trichrome for Muscle, Cartilage and Collagen

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

Protocol

Kostowiecki's Trichrome

for Muscle, Cartilage and Collagen

steps

materials

print

Materials

A red nuclear stain
Solution A
Material Amount
Aniline blue 0.06 g
Orange G 0.2 g
Phosphomolybdic acid 1 g
Distilled water 100 mL

Material	Amount
Aniline blue	0.06	g
Orange G	0.2	g
Phosphomolybdic acid	1	g
Distilled water	100	mL

Preparation of Solution A

Add both dyes to the water.
Bring to the boil for three minutes.
Add the phosphomolybdic acid while hot.
Cool 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 a red nuclear stain.
Place into the staining solution until darkly stained, 30-120 minutes.
Rinse with distilled water.
Place in 95% ethanol for 1 minute.
Dehydrate with absolute ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – red
Muscle – orange
Cartilage – blue
Collagen – blue-green

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:
Kostowiecki, (1932)
Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik,
v. 49, pp. 337

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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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

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

Material	Amount
Acid fuchsin	1	g
Distilled water	100	mL

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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Goldner’s Trichrome for Muscle and Collagen

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

Protocol

Goldner's Trichrome

for Muscle and Collagen

steps

materials

print

Materials

Weigert’s iron hematoxylin or equivalent
Solution A
Material Variation 1 Variation 2 Variation 3
Ponceau 2R 0.7 g 0.75 g –
Acid fuchsin 0.3 g 0.25 g –
Azophloxine – 0.1 g 5 g
Acetic acid, glacial 2 mL 2 mL 2 mL
Distilled water 1 L 1 L 1 L
Solution B
Material Amount
Acetic acid, glacial 10 mL
Distilled water 1 L
Solution C
Material Amount
Orange G 20 g
Phosphomolybdic acid 40 g
Distilled water 1 L
Solution D
Material Amount
Light green SF 2 g
Acetic acid, glacial 2 mL
Distilled water 1 L

Material	Variation 1	Variation 2	Variation 3
Ponceau 2R	0.7	g	0.75	g	–
Acid fuchsin	0.3	g	0.25	g	–
Azophloxine	–		0.1	g	5	g
Acetic acid, glacial	2	mL	2	mL	2	mL
Distilled water	1	L	1	L	1	L

Material	Amount
Acetic acid, glacial	10	mL
Distilled water	1	L

Material	Amount
Orange G	20	g
Phosphomolybdic acid	40	g
Distilled water	1	L

Material	Amount
Light green SF	2	g
Acetic acid, glacial	2	mL
Distilled water	1	L

Tissue Sample

Protocol

Bring sections to water via xylene and ethanol.
Stain nuclei with Weigert’s iron hematoxylin, or equivalent.
Place into one of the variants of solution A for 5 minutes.
Rinse with solution B.
Place into solution C until collagen is decolorised.
Rinse with solution B.
Place into solution D for 5 minutes.
Place into solution B for 5 minutes.
Blot.
Dehydrate rapidly with absolute ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

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

Safety Note

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

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.

March 30, 2023

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Modified Yellowsolve for General Oversight

By Carmen WongProtocols, Stain Type, Trichrome Staining, Yellowsolve Staining

Protocol

Modified Yellowsolve

for General Oversight

steps

materials

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Materials

Hemalum
Trichlorethylene
Solution A
Material Amount
Phloxine B 0.5 g
Calcium chloride 0.5 g
Distilled water 100 mL
Solution B
Material Amount
Luxine pure yellow to saturation
2-Ethoxyethanol 50 mL
Ethyl phosphate 50 mL

Material	Amount
Phloxine B	0.5	g
Calcium chloride	0.5	g
Distilled water	100	mL

Material	Amount
Luxine pure yellow	to	saturation
2-Ethoxyethanol	50	mL
Ethyl phosphate	50	mL

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are likely suitable. It should be noted, however, that the authors of this method favoured extended fixation in formal sublimate of up to 10 days for fibrin. This fixative is now deprecated due to its mercuric chloride content. With other staining methods, pretreatment of sections with Bouin’s fluid for an hour at 60°C can compensate for the lack of mercury fixation to a large degree.

Lendrum says, “If … the time of differentiation is kept short the result is comparable to the best examples of the old Masson’s erythrosin-saffron technique.” Masson’s erythrosin-saffron is the same technique as the HPS (hematoxylin-phloxine-saffron) using phloxine instead of erythrosin.

Protocol

Dewax sections with xylene.
Place in trichlorethylene for 24 to 48 hours.
Bring sections to water.
Stain nuclei with hemalum, differentiate and blue.
Wash well with water.
Place in solution A for 30 minutes.
Rinse with 2-ethoxyethanol.
Differentiate with solution B, controlling microscopically, until collagen is yellow.
Rinse with 2-ethoxyethanol.
Clear in xylene and mount with a synthetic resinous medium.

Expected Results

Nuclei – blue
Muscle – red
Background – yellow

Notes

Stop differentiation when collagen is yellow and muscle is still red.
2-Ethoxyethanol is also known as cellosolve or ethylene glycol monoethyl ether, and has the formula CH₃CH₂OCH₂CH₂OH.
Trichlorethylene has the formula ClHC=CCl₂ or C₂HCl₃
Trichlorethylene should be used in a fume hood.

Safety Note

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

References

Lendrum A C, Fraser D S, Slidders W and Henderson R. (1962)
Studies on the character and staining of fibrin.
Journal of clinical pathology, v. 15, p. 401.

March 30, 2023

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Picro-Mallory for Fibrin – Shorter Version

By Carmen WongFibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, Multi-Step

Protocol

Picro-Mallory

for Fibrin – Shorter Version

steps

materials

print

This version of the picro-Mallory is relatively uncomplicated and is suitable for a routine pathology laboratory. Proper fixation is still essential and minimalist formalin fixation with quick processing should be avoided as it will give disappointing results with poorly stained erythrocytes. Originally, extended mercury fixation, thorough processing, degreasing and refixing in picro-mercuric-ethanol were specified. Although fixation in formol sublimate (or B5) is preferred for this method as well, adequate results can be obtained with overnight formalin fixation and overnight processing. Refixation of sections in Bouin’s fluid at 56°C for an hour or so overcomes some of the deficiencies in fixation, but results are still inferior to those obtained by the full procedure.

Materials

An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
Picro-orange
Material Amount
Picric acid, sat. in 80% ethanol 100 mL
Orange G 0.2 g
Yellow differentiator
Material Amount
Picro-orange 30 mL
Ethanol, 80% 70 mL
Red stain
Material Amount
Acid fuchsin 1 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Red differentiator
Material Amount
Phosphotungstic acid 1 g
Distilled water 100 mL
Blue stain
Material Amount
Methyl blue 2 g
Acetic acid, glacial 2 mL
Distilled water 98 mL

Material	Amount
Picric acid, sat. in 80% ethanol	100	mL
Orange G	0.2	g

Material	Amount
Picro-orange	30	mL
Ethanol, 80%	70	mL

Material	Amount
Acid fuchsin	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Material	Amount
Phosphotungstic acid	1	g
Distilled water	100	mL

Material	Amount
Methyl blue	2	g
Acetic acid, glacial	2	mL
Distilled water	98	mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate (or B5) overnight. Paraffin process overnight. Avoid rapid fixation with formalin and short processing, as this produces tissues that stain poorly even with the secondary fixation specified. Sections should be 3-5 µ thick.

Protocol

Bring sections to water via xylene and ethanol.
Remove mercury pigment with the iodine-thiosulphate sequence.
Place into Bouin’s fluid at 56°C for 1 hour.
Rinse well in distilled water.
Stain nuclei with an acid resistant nuclear stain.
Rinse with 95% ethanol.
Place in picro-orange for 2 minutes.
Without rinsing, place in the red stain for 5 minutes.
Rinse with distilled water.
Dip into yellow differentiator.
Rinse in distilled water.
Differentiate with the red differentiator for 5 minutes.
Rinse with distilled water.
Place in the blue stain for 2 minutes.
Rinse briefly with 1% aqueous acetic acid.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Fibrin – clear red
Fibrinoid – varies from red to orange
Erythrocytes – orange
Connective tissue – blue

Notes

If possible, the extended fixation and slow processing specified for the longer version should be used. Degreasing and refixation will also improve results if time allows.

Safety Note

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

References

Culling C.F.A., (1974)
Handbook of histopathological and histochemical techniques Ed. 3
Butterworth, London, UK.

March 30, 2023

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