Picro-Mallory

for Fibrin – Long Version

steps

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Lendrum, Fraser and others published several fibrin stains, the most well known being the Picro-Mallory. There are several variants of the method, some more complicated than others.

See this alternative protocol for a shorter version.

With this trichrome stain full and complete fixation is absolutely essential. Minimalist formalin fixation and quick processing will give poorly stained erythrocytes with red or orange tinges instead of the yellow they should have. Lendrum and coworkers specified about seven days fixation in formal sublimate followed by processing thoroughly, sectioning, degreasing with trichlorethylene for 48 hours, then refixing in picro-mercuric-alcohol. Adequate results can be obtained with thorough formalin fixation (24 hours), thorough processing (overnight), sectioning, followed by refixing in Bouin’s fluid at 56°C for an hour or so. However, results are inferior to those obtained by the full procedure. It is also a method where experience is required for optimal results.

Materials

An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the
celestine blue-hemalum sequence.
Yellow mordant
Material Amount
Picric acid, sat. in 80% ethanol 100 mL
Orange G 0.2 g
Lissamine yellow 0.2 g
Stock differentiator
Material Amount
Picric acid 2.5 g
Ethanol, 95% 100 mL
Phosphotungstic acid 25 g
Dissolve ingredients and filter.
Red differentiator
Material Amount
Stock differentiator 40 mL
Ethanol, 95% 20 mL
Distilled water 90 mL
Blue differentiator
Material Amount
Stock differentiator 10 mL
Distilled water 90 mL
Red stain
Material Option 1 Option 2 Option 3
Acid fuchsin 1 g – 0.4 g
Lissamine fast red – – 0.2 g
Biebrich scarlet – 1 g –
Acetic acid, glacial 1 mL 1 mL 1 mL
Distilled water 99 mL 99 mL 99 mL
Blue stain
Material Amount
Methyl blue 1 g
Acetic acid, glacial 1 mL
Distilled water 99 mL

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

Material	Amount
Picric acid	2.5	g
Ethanol, 95%	100	mL
Phosphotungstic acid	25	g

Material	Amount
Stock differentiator	40	mL
Ethanol, 95%	20	mL
Distilled water	90	mL

Material	Amount
Stock differentiator	10	mL
Distilled water	90	mL

Material	Option 1	Option 2	Option 3
Acid fuchsin	1	g	–	0.4	g
Lissamine fast red	–	–	0.2	g
Biebrich scarlet	–	1	g	–
Acetic acid, glacial	1	mL	1	mL	1	mL
Distilled water	99	mL	99	mL	99	mL

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

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate for one week. They should be paraffin processed with a schedule that thoroughly and completely dehydrates, then thoroughly cleared with xylene and infiltrated with paraffin wax. This process would normally take 48 hours or longer. Err on the side of completeness, and do not attempt to shorten the process. Avoid rapid fixation and overnight processing, especially with formalin fixatives, as this produces tissues that stain poorly even with secondary fixation such as Bouin’s fluid at 56°C for an hour. Sections should be 3-5 µ thick.

Protocol

Bring sections to water via xylene and ethanol.
Remove mercury pigment with the iodine-thiosulphate sequence.
Stain nuclei with an acid resistant nuclear stain.
Place in yellow mordant for 2-3 minutes.
Wash in distilled water until only erythrocytes are yellow.
Place in the red stain for 5-10 minutes.
Rinse with 1% aqueous acetic acid.
Differentiate with the red differentiator until fibrin is prominent microscopically.
Rinse well in distilled water.
Place in the blue stain for 5 minutes.
Rinse briefly with 1% aqueous acetic acid.
Place in the blue differentiator for 1-2 minutes.
Biefly rinse with 1% aqueous acetic acid.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Fibrin – clear red
Erythrocytes – yellow
Connective tissue – blue

Notes

The most common dye combination is acid fuchsin and methyl blue.
Adequate results may be obtained with 24-48 hours fixation in neutral buffered formalin, overnight paraffin processing and secondary fixation of sections with Bouin’s picro-formol-acetic for 1 hour at 56°C.
Good results are obtained with the fixation and processing recommended in the text.
Optimal results are obtained with the fixation and processing recommended in the text, followed by degreasing and secondary fixation in picro-mercuric ethanol.
Replace step 1 with the following:
1. Dewax sections with xylene.
2. Place in trichlorethylene in a sealed container at 56°C for 48 hours.
3. Rinse sections well with absolute ethanol.
4. Place in picro-mercuric-ethanol for 24 hours.
5. Wash sections with water, and continue from step 2.

Safety Note

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

References

Lendrum, A. C., et. al. (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

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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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Yellowsolve I for Fibrin

By Carmen WongFibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Yellowsolve Staining

Protocol

Yellowsolve I

for Fibrin

steps

materials

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Materials

Celestine blue-hemalum nuclear stain, or similar.
Trichlorethylene
Tetrachlorethylene
Solution A
Material Amount
Phloxine B 0.5 g
Calcium chloride 0.5 g
Distilled water 100 mL
Solution B
Material Amount
Lissamine flavine FF to saturation
2-Ethoxyethanol 50 mL
Ethyl phosphate 50 mL
Optional – an elastic fibre stain: Orcein or Weigert’s iron resorcin fuchsin, or variant.

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

Material	Amount
Lissamine flavine FF	to saturation
2-Ethoxyethanol	50	mL
Ethyl phosphate	50	mL

Tissue Sample

5µ 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.

Protocol

Dewax sections with xylene.
Place in trichlorethylene for 24 to 48 hours.
Bring sections to water.
Stain elastic fibres if desired.
Stain nuclei with the celestine blue-hemalum sequence.
Wash well with water.
Place in solution A for 30 minutes.
Rinse with 2-ethoxyethanol then with tetrachlorethylene.
Place into tetrachlorethylene overnight.
Rinse with 2-ethoxyethanol.
Differentiate with solution B, controlling microscopically.
Rinse with 2-ethoxyethanol.
Clear in xylene and mount with a synthetic resinous medium.

Expected Results

Fibrin – red
Background – yellow
Nuclei – black
Acidophil cytoplasmic inclusions – red

Notes

2-Ethoxyethanol is also known as cellosolve or ethylene glycol monoethyl ether, and has the formula CH₃CH₂OCH₂CH₂OH.
Tetrachlorethylene has the formula Cl₂C=CCl₂ or C₂Cl₄
Trichlorethylene has the formula ClHC=CCl₂ or C₂HCl₃
Both trichlorethylene and tetrachlorethylene 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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Foot’s Impregnation for Reticulin

By Carmen WongMetal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type

Protocol

Foot's Impregnation

for Reticulin

steps

materials

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Materials

Material	Variation I	Variation II	Variation III	Variation IV
Lugol’s iodine	+	+	+	+
Sodium thiosulphate, 1% aqu.	+	+	+	+
Sodium thiosulphate, 5% aqu.	+	+	+	+
Potassium permanganate, 0.25% aqu.	+	+	+	+
Oxalic acid, 5% aqu.	+	+	+	+
Strong ammonium hydroxide (s.g. 0.88)	+	+	+	+
Ammonium hydroxide, 0.1% aqu	–	+	–	–
Foot’s tannin sol.	–	+	–	–
Silver nitrate, aqu.	2% & 10%	1% & 10%	10%	10%
Formalin, aqu.	5%	20%	20%	1%
Yellow gold chloride, aqu.	1%	–	0.2%	0.2%
Foot’s gold sublimate	–	+	–	–
Alkali, aqu.	NaOH 40%	KOH 40%	LiC0₃ sat.	Na₂CO₃
Weigert’s 1903 iron hematoxylin	+	–	–	–
Harris alum hematoxylin	–	–	+	–
van Gieson’s picro-fuchsin	+	–	+	–
Neutral red, 1% aqu.	–	–	–	+

Lugol’s Iodine (see note)

Material	Amount
Iodine	6	g
Potassium iodide	4	g
Distilled Water	100	mL

Mix the iodine and potassium iodide in a 200 mL flask. Add 10 mL of the water. When the iodine has dissolved make up to 100 mL with distilled water.

Foot’s gold sublimate

Material	Amount
Yellow gold chloride	0.2	g
Mercuric chloride	0.5	g
Distilled Water	100	mL

Foot’s tannin solution

Material	Amount
Tannic acid	0.15	g
Ammonium bromide	3.5	g
Strong formalin	5	mL
Distilled Water	100	mL

Preparation of Foot’s Ammoniacal Silver

Variation I

Place 25 mL of 10% silver nitrate in a flask.
Add 1 mL of 40% sodium hydroxide.
Add ammonium hydroxide by drops until the precipitate is almost dissolved.
Dilute to 100 mL with distilled water.

Variation II

Place 10 mL of 1% silver nitrate in a flask.
Add 0.1 mL of 40% potassium hydroxide.
Add ammonium hydroxide by drops until the precipitate is almost dissolved.
Dilute to 100 mL with distilled water.

Variation III

Place 10 mL of 10% silver nitrate in a flask.
Add 10 mL of saturated aqueous lithium carbonate.
Let the precipitate settle, then decant the supernatent.
Wash, allow to settle, and decant several times.
Add 25 mL distilled water to the precipitate.
Add ammonium hydroxide by drops until the precipitate is almost dissolved.
Dilute to 100 mL with distilled water and filter.

Variation IV

Place 10 mL of 10% silver nitrate in a flask.
Add 40 mL of aqueous sodium carbonate (see note for concentration).
Let the precipitate settle, then decant the supernatent.
Wash, allow to settle and decant several times.
Add ammonium hydroxide by drops until the precipitate is almost dissolved.

Tissue Sample

5 µ paraffin sections of Zenker fixed tissue are suitable. Other fixatives may be satisfactory. Carleton & Leach say that Var IV is suitable for 10% formalin, Zenker, Bouin or Helly fixed tissue. In all cases a section adhesive is recommended.

Protocol

Step		Variation I	Variation II	Variation III	Variation IV
1	Bring sections to water via xylene and ethanol (all variations).
2	Place in Lugol’s iodine.	5 min	5 min	5 min	5 min
3	Bleach in 1% sodium thiosulphate.	30 sec	30 sec	30 sec	30 sec
4	Oxidise in 0.25% potassium permanganate.	5 min	5 min	5 min	5 min
5	Bleach with 5% oxalic acid.	10 min	10 min	10 min	10 min
6	Wash well with tap water.	5 min	5 min	5 min	5 min
7	Rinse with distilled water.	5 sec	5 sec	5 sec	5 sec
8	Place in 2% silver nitrate.	48 hrs	—	—	—
	Place in Foot’s tannin solution at 37°C.	—	15 min	—	—
	Place in 0.1% ammonium hydroxide.	—	30 sec	—	—
9	Rinse with distilled water.	5 sec	5 sec	—	—
10	Place in Foot’s ammoniacal silver solution (Variation I).	30 min	—	—	—
	Place in Foot’s ammoniacal silver solution (Variation II), 2 changes, each for:	—	5 min	—	—
	Place in Foot’s ammoniacal silver solution (Variation III) at 45°C.	—	—	15 min	—
	Place in Foot’s ammoniacal silver solution (Variation IV) at 45°C.	—	—	—	15 min
11	Rinse with distilled water.	5 sec	5 sec	5 sec	—
12	Reduce with 10% formalin.	30 min	—	—	—
	Reduce with 20% formalin.	—	3 min	2 min	—
	Flood with 1% formalin twice.	—	—	—	10 sec
13	Rinse with distilled water.	—	—	5 sec	—
	Wash off with tap water.	—	—	—	5 sec
14	Tone with 1% yellow gold chloride.	1 hr	—	—	—
	Tone with Foot’s gold sublimate.	3 min	—	—	—
	Tone with 0.2% yellow gold chloride.	—	—	2 min	2 min
15	Wash with distilled water.	1 min	1 min	—	—
	Wash with tap water.	—	—	—	2 min
16	Place in 5% sodium thiosulphate.	—	3 min	2 min	2 min
17	Wash with tap water.	5 min	5 min	5 min	5 min
18	Place in Weigert’s iron hematoxylin.	1 min	—	—	—
	Place in Harris’ alum hematoxylin.	—	—	3 min	—
	Place in neutral red.	—	—	—	1 min
19	Wash with tap wate.	5 min	5 min	5 min	1 min
20	Place in van Gieson’s picro-fuchsin.	30 sec	—	45 sec	—
22	Dehydrate with ethanol, clear with xylene and mount in a resinous medium (all variations).

Expected Results

Reticulin fibres – black
Nuclei – as counterstained
Background – grey

Notes

Ensure that the ammonium hydroxide is fresh and full strength. Keep well stoppered when not in use. After removing the amount required, immediately restopper the bottle.
Improperly made ammoniacal silver solutions can affect the quality of the impregnation. There should be a faint, persistent opalescence, with only a faint smell of ammonia.
The formula for Lugol’s iodine may be an error. The amounts may have been reversed,
i.e. It may be 4 g iodine and 6 g potassium iodide. Please refer to the page for
Lugol’s iodine and compare Gray and Lee. The details for these methods are from Gray.
The instructions for ammoniacal silver solution Var IV says to add 40 mL of sodium carbonate to the silver nitrate solution. The sodium carbonate should be made as a 5% solution of the anhydrous salt or as an 11% solution of the dodecahydrate (12H₂O).
1%, 10% and 20% formalin solutions are made by diluting strong formalin appropriately with water, i.e.:
- 1% is 1 mL strong formalin plus 99 mL water,
- 10% is 10 mL strong formalin plus 90 mL water, and
- 20% is 20 mL strong formalin plus 80 mL water.

Safety Note

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

References

Gray, Peter. (1954), (for Var I, II & III)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Carleton, H M, and Leach, E H, (1938), (for Var IV)
Histological technique., Ed. 2.
Oxford University Press, London, England.

March 30, 2023

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Gridley’s Stain for Fungi

By Carmen WongAldehydes, Chromic Acid-Schiff Reaction, Protocols, Schiff's Reagent Reactions, Stain Target, Stain Type

Protocol

Gridley's Stain

for Fungi

steps

materials

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Materials

Schiff’s reagent
Aldehyde fuchsin
Chromic acid
Material Amount
Chromium trioxide 5 g
Distilled water 500 mL
Bleach
Material Amount
Sodium metabisulfite 5 g
Distilled water 500 mL
Metanil yellow
Material Amount
Metanil yellow 1 g
Distilled water 400 mL
Acetic acid, glacial 2 drops

Material	Amount
Chromium trioxide	5	g
Distilled water	500	mL

Material	Amount
Sodium metabisulfite	5	g
Distilled water	500	mL

Material	Amount
Metanil yellow	1	g
Distilled water	400	mL
Acetic acid, glacial	2	drops

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable.

Protocol

Bring sections to water via xylene and ethanol.
Place in chromic acid for 1 hour.
Wash well with tap water.
Treat with the metabisulfite bleach for 1 minute.
Wash well with tap water.
Rinse with distilled water.
Place in Schiff’s reagent for 20 minutes.
Wash well with tap water.
Rinse with 70% ethanol.
Place in aldehyde fuchsin 30 minutes.
Rinse off excess with 95% ethanol.
Wash well with tap water.
Counterstain with metanil yellow for 1 minute.
Rinse well with distilled water.
Dehydrate, clear and mount in a resinous medium.

Expected Results

Fungi – purple
Background – yellow

Notes

At step 2, a 10% solution of chromic acid applied for 10 minutes will give similar results.

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.
Humason, G. L., (1967)
Animal tissue techniques, Ed. 2
W. H. Freeman and Company, San Francisco, Ca, USA

March 30, 2023

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French’s Stain for Elastic Fibres

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

Protocol

French's Stain

for Elastic Fibres

steps

materials

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Materials

Weigert’s iron hematoxylin or equivalent
Van Gieson’s picro-fuchsin
French’s solution
Material Amount
Basic fuchsin 1 g
Crystal violet 1 g
Resorcin 4 g
Ferric chloride, 30% aqu. 25 mL
Ethanol 95% 200 mL
Distilled water 200 mL
Hydrochloric acid, conc. 4 mL
Preparation
1. Add the dye and resorcin to the water in an oversize flask.
2. Bring to the boil, and add the ferric chloride.
3. Boil for 5 minutes.
4. Cool and filter.
5. Dry the filter paper and beaker. Place the precipitate and filter paper back into the flask.
6. Add the ethanol and heat carefully until the precipitate dissolves.
7. Add the hydrochloric acid.
8. Restore to 200 mL with 95% ethanol.

Material	Amount
Basic fuchsin	1	g
Crystal violet	1	g
Resorcin	4	g
Ferric chloride, 30% aqu.	25	mL
Ethanol 95%	200	mL
Distilled water	200	mL
Hydrochloric acid, conc.	4	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.
Place into French’s solution until adequately stained (may be overnight).
Wash with 95% ethanol to remove excess solution.
Differentiate with 1% acid alcohol.
Wash in water.
Counterstain with iron hematoxylin and van Gieson.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Elastic fibres – dark blue-green
Collagen – red
Cytoplasm – yellow
Nuclei – blue

Notes

This is Weigert’s iron resorcin fuchsin using crystal violet and basic fuchsin instead of basic fuchsin alone.
It is usually recommended that the basic fuchsin should not be of the type that produces good Schiff’s reagent. That is, the basic fuchsin should contain more rosanilin than pararosanilin.
The ferric chloride solution should be freshly made.

Safety Note

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

References

Histological demonstration techniques, (1974)
Cook, H C.
Butterworths, London, England

March 30, 2023

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Fullmer & Lillie’s Orcinol-New Fuchsin for Elastic Fibres

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

Protocol

Fullmer & Lillie's Orcinol-New Fuchsin

for Elastic Fibres

steps

materials

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Materials

- Weigert’s iron hematoxylin or equivalent
- Van Gieson’s picro-fuchsin
- Orcinol-New Fuchsin solution
  Material Amount
  New fuchsin 2 g
  Orcinol 4 g
  Ferric chloride, 30% aqu. 50 mL
  Distilled water 200 mL
  Ethanol 95% 100 mL
  Preparation
  1. Add the dye and orcinol to the water in an oversize flask.
  2. Boil for 5 minutes.
  3. Add the ferric chloride.
  4. Boil for a further 5 minutes.
  5. Cool and filter.
  6. Dry the filter paper and beaker. Place the precipitate and filter paper back into the flask.
  7. Add the ethanol and heat carefully until the precipitate dissolves.
  8. Restore to 100 mL with 95% ethanol.

Material	Amount
New fuchsin	2	g
Orcinol	4	g
Ferric chloride, 30% aqu.	50	mL
Distilled water	200	mL
Ethanol 95%	100	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.
Place into orcinol-new fuchsin solution for 15 minutes at 37°C.
Wash with 3 changes of 70% ethanol for 5 minutes each.
Counterstain with iron hematoxylin and van Gieson.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Elastic fibres – deep violet
Collagen – red
Cytoplasm – yellow
Nuclei – black

Notes

Fullmer and Lillie specified 25mL USP IX Liquor Ferri Chloridi, or a solution of 15.5g ferric chloride (FeCl₃.6H₂O) made up to 25 mL with water, i.e. 25mL of a 62% solution. In either case, it should be fresh.

Safety Note

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

References

Fullmer, H M and Lillie, R D, (1956).
A selective stain for elastic tissue (orcinol-new fuchsin).,
Stain Technologyl, v 31, page 27-29.
Lillie, R D and Fullmer, H M, (1976).
Histopathological technic and practical histochemistry, Ed. 4. pp. 714.
McGraw-Hill, New York, USA.

March 30, 2023

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Hart’s Stain for Elastic Fibres

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

Protocol

Hart's Stain

for Elastic Fibres

steps

materials

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Materials

Weigert’s iron hematoxylin or equivalent
Van Gieson’s picro-fuchsin
Hart’s solution
Material Amount
Weigert’s iron resorcin fuchsin 1 volume
1% hydrochloric acid in 70% ethanol 9 volumes

Material	Amount
Weigert’s iron resorcin fuchsin	1	volume
1% hydrochloric acid in 70% ethanol	9	volumes

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.
Place into Hart’s solution overnight.
Wash with 95% ethanol to remove excess solution.
Differentiate with 1% acid alcohol.
Wash with tap water.
Counterstain with iron hematoxylin and van Gieson.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Elastic fibres – blue black
Collagen – red
Cytoplasm – yellow
Nuclei – blue

Notes

This modification should be used if poor staining is obtained using Weigert’s method.
The optimum dilution may be more or less than given.

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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Humberstone’s Stain for Elastic Fibres

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

Protocol

Humberstone's Stain

for Elastic Fibres

steps

materials

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Materials

Weigert’s iron hematoxylin or equivalent
Van Gieson’s picro-fuchsin
Potassium permanganate, 0.25% aqu., for the Mallory bleach
Humberstone’s solution
Material Amount
Victoria blue 4R 1 g
Ethyl violet 1 g
Resorcin 4 g
Dextrin 0.5 g
Ferric chloride, 30% aqu. 25 mL
Ethanol, 95% 150 mL
Phenol 10 g
Hydrochloric acid, conc. 4 mL
Distilled water 200 mL

Material	Amount
Victoria blue 4R	1	g
Ethyl violet	1	g
Resorcin	4	g
Dextrin	0.5	g
Ferric chloride, 30% aqu.	25	mL
Ethanol, 95%	150	mL
Phenol	10	g
Hydrochloric acid, conc.	4	mL
Distilled water	200	mL

Preparation

In an oversized flask, add the dyes to the water, and boil to dissolve.
Add the resorcin, dextrin and ferric chloride. Boil for 3 minutes. Cool and filter.
Place the precipitate and filter paper back into the original flask. Add 100 ml of 95% ethanol and ently for 15 minutes.
Cool and filter.
Make up the volume to 350 mL with 95% ethanol.
Add the phenol and hydrochloric acid.

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.
Do a Mallory bleach with 0.25% potassium permanganate for ten minutes.
Rinse with 95% ethanol.
Place into Humberstone’s solution overnight.
Wash with 95% ethanol to remove excess solution.
Wash in water.
Counterstain with iron hematoxylin and van Gieson.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Elastic fibres – blue-black
Nuclei – black
Cytoplasm – yellow
Collagen – red

Notes

The authors state that buffered formalin is an unsuitable fixative. I have not found this so and have used this method regularly with such tissue. It may be that the modern practice of minimalist fixation limits the effect.
As the solution ages, the demonstration of elastic fibres improves in both quality and speed. When the solution is a few years old elastic is demonstrated within about 4 hours, although overnight is generally
preferable.
The solution is stable for more than ten years. Due to the improvement in staining as the solution ages, it is suggested that a litre, or more, be made at a time.
The authors suggest as suitable counterstains, iron hematoxylin and van Gieson, Masson’s trichrome or MSB (with green collagen).

Safety Note

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

References

Humberstone, G. C. W., and Humberstone, F. D., (1969)
An Elastic Tissue Stain
Journal of Medical Laboratory Technology, V. 26, No 2, pp. 99.

March 30, 2023

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Miller’s Stain for Elastic Fibres

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

Protocol

Miller's Stain

for Elastic Fibres

steps

materials

print

Materials

Weigert’s iron hematoxylin or equivalent
Van Gieson’s picro-fuchsin
Potassium permanganate, 0.25% aqu., for the Mallory bleach
Miller’s solution
Material Amount
Victoria blue 4R 1 g
New fuchsin 1 g
Crystal violet 1 g
Resorcin 4 g
Dextrin 1 g
Ferric chloride, 30% aqu. 50 mL
Distilled water 200 mL
Ethanol, 95% 200 mL
Hydrochloric acid, conc. 2 mL

Material	Amount
Victoria blue 4R	1	g
New fuchsin	1	g
Crystal violet	1	g
Resorcin	4	g
Dextrin	1	g
Ferric chloride, 30% aqu.	50	mL
Distilled water	200	mL
Ethanol, 95%	200	mL
Hydrochloric acid, conc.	2	mL

Preparation

In an oversized flask heat the water and add the three dyes.
When dissolved, add the resorcin, dextrin and ferric chloride in the order given.
Boil for 5 minutes, then filter while hot.
Place the precipitate and filter paper back into the original flask, add the 95% ethanol and simmer gently for 20 minutes.
Cool and filter.
Restore the volume to 200 mL with 95% ethanol, then add 2 mL of concentrated hydrochloric acid.

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.
Do a Mallory bleach with 0.25% potassium permanganate for ten minutes.
Rinse with 95% ethanol.
Place into Miller’s solution up to three hours.
Wash with 95% ethanol to remove excess solution.
Wash in water.
Counterstain with iron hematoxylin and van Gieson.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Elastic fibres – black
Nuclei – black
Cytoplasm – yellow
Collagen – red

Notes

Three hours is recommended, although staining may be adequate after 90 minutes.
Dilute with an equal quantity of 95% ethanol for overnight staining.
The staining solution is stable for years.

Safety Note

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

References

Miller, P.J., (1971)
Journal of Medical Laboratory Technology, V. 28
Ellis, R.,
Miller’s Elastic Staining Protocol
IHC World

March 30, 2023

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