Musto's Hematoxylin Scarlet-Saffron

for Elastic

steps

materials

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Materials

Stock hematoxylin

Material	Amount
Hematoxylin	2	g
Ethanol, 95%	100	mL

Stock ferric chloride

Material	Amount
Ferric chloride, hexahydrate	12.4	g
Distilled water	500	mL
Hydrochloric acid, concentrated	5	mL

Stock iodine

Material	Amount
Potassium iodide	4	g
Iodine	2	g
Distilled water	100	mL

Working solution

Material	Amount
Stock hematoxylin	30	mL
Stock ferric chloride	20	mL
Stock iodine	10	mL

Tungstophosphoric acid

Material	Amount
Tungstophosphoric acid	5	g
Distilled water	100	mL

Acetic acid

Material	Amount
Acetic acid, glacial	1	mL
Distilled water	99	mL

Woodstain scarlet

Material	Amount
Woodstain scarlet, 1% aqu.	50	mL
Tungstophosphoric acid	2	g

Prepare fresh.

Alcoholic saffron solution

Material	Amount
Saffron	1	g
Ethanol, absolute	100	mL

Extract the saffron in ethanol at 56°C for 24 hours, shaking occasionally. Cool and filter.

Tissue Sample

A 10% Formalin variant is suitable. Paraffin sections at 5µ are preferred.

Protocol

Bring sections to water via xylene and ethanol.
Place sections in Bouin’s fluid at 60°C for 1 hour.
Remove the yellow colouration with running tap water.
Place in working solution for 45 minutes.
Wash well with tap water, then rinse with distilled water.
Place into the woodstain scarlet solution for 5 minutes.
Rinse well with distilled water.
Differentiate in tungstophosphoric acid solution for 5 minutes.
Transfer directly to acetic acid solution for 5 minutes.
Dehydrate with 95% ethanol for 1 minute.
Place in absolute ethanol, two changes, for 1 minute each.
Place in alcoholic saffron solution for 5-10 minutes.
Place in absolute ethanol, two changes, for 1 minute each.
Clear with xylene and mount with a resinous medium

Expected Results

Nuclei – black
Elastic fibres – black
Muscle – pale red
Collagen – yellow

Notes

The original paper also gave a procedure using a Van Gieson counterstain.
This is a modification of Verhöeff’s hematoxylin which does not require differentiating.

Safety Note

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

References

Musto, Linda, (1981),
Improved iron-hematoxylin stain for elastic fibers.,
Stain Technology, v 56, page 185-7.

March 30, 2023

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Musto’s Hematoxylin for Elastic Fibres

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

Protocol

Musto's Hematoxylin

for Elastic Fibres

steps

materials

print

Materials

Stock hematoxylin

Material	Amount
Hematoxylin	2	g
Ethanol, 95%	100	mL

Stock ferric chloride

Material	Amount
Ferric chloride, hexahydrate	12.4	g
Distilled water	500	mL
Hydrochloric acid, concentrated	5	mL

Stock iodine

Material	Amount
Potassium iodide	4	g
Iodine	2	g
Distilled water	100	mL

Thiosulphate solution

Material	Amount
Sodium thiosulphate	5	g
Distilled water	100	mL

Working solution

Material	Amount
Stock hematoxylin	30	mL
Stock ferric chloride	20	mL
Stock iodine	10	mL

Tissue Sample

A 10% Formalin variant is suitable. Paraffin sections at 5µ are preferred.

Protocol

Bring sections to water via xylene and ethanol.
Place sections in Bouin’s fluid at 60°C for 1 hour.
Remove the yellow colouration with running tap water.
Place in working solution for 45 minutes.
Wash well with tap water, then rinse with distilled water.
Place into thiosulphate solution for 2 minutes to remove iodine discolouration.
Wash well with tap water, then rinse with distilled water.
Counterstain with Van Gieson.
Dehydrate with absolute ethanol.
Clear with xylene and mount with a resinous medium

Expected Results

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

Notes

The original paper recommended counterstaining with a modification of the HPS.
The treatment with Bouin’s solution is likely included to improve an HPS stain. It may be possible to eliminate it for counterstaining with Van Gieson.
This is a modification of Verhöeff’s hematoxylin which does not require differentiating, although the Van Gieson counterstain may diminish the intensity of the stain.

Safety Note

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

References

Musto, Linda, (1981),
Improved iron-hematoxylin stain for elastic fibers.,
Stain Technology, v 56, page 185-7.

March 30, 2023

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Verhoeff’s Iron Hematoxylin for Elastic

By Carmen WongDye Type, Elastic Fibers, Hematoxylin and Eosin Staining, Iron Hematoxylin, Mordanted Hematoxylin, Protocols, Stain Target, Stain Type

Protocol

Verhoeff's Iron Hematoxylin

for Elastic

steps

materials

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Materials

Stock Verhoeff A

Material	Amount
Hematoxylin	1	g
Ethanol, absolute	20	mL

Stock Verhoeff B

Material	Amount
Ferric chloride	10	g
Distilled water	100	mL

Stock Verhoeff C

Material	Amount
Potassium iodide	4	g
Iodine	2	g
Distilled water	100	mL

Working solution

Material	Amount
Stock solution A	20	mL
Stock solution B	8	mL
Stock solution C	8	mL

Differentiator

Material	Amount
Stock solution B	10	mL
Distilled water	40	mL

Tissue Sample

A 10% Formalin variant is suitable. Paraffin sections at 5µ are preferred.

Protocol

Bring sections to water via xylene and ethanol.
Place in working Verhoeff’s solution for 15 minutes.
Wash well with tap water to remove all excess hematoxylin.
Differentiate until the elastic fibres are satisfactory. This should be controlled microscopically based on the appearance of fibres in the area of interest.
Rinse well with tap water.
Place into 95% ethanol for 5 minutes to remove iodine discolouration.
Wash well with tap water to remove all residual chemicals.
Counterstain with Van Gieson.
Dehydrate with absolute ethanol.
Clear with xylene and mount with a resinous medium

Expected Results

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

Notes

The alcoholic hematoxylin solution should be freshly made. It does not need to have been ripened as ferric chloride is an oxidising agent. Some technologists keep pre-weighed aliquots of 1 gram hematoxylin and dissolve it in 20 mL ethanol as needed.
The working solution should be made just before use and allowed to stand for five minutes to ripen.
While this is a popular elastic stain, it is difficult to stain both coarse fibres and fine fibres optimally in a single section. If coarse fibres are well stained, the finest fibres may be completely decolourised, and if fine fibres are optimally stained, the coarse fibres are underdifferentiated. Ensure differentiation is optimised for the fibres of interest.
If Van Gieson is used as a counterstain, the elastic fibres should be very slightly underdifferentiated as picric acid will also remove some hematoxylin staining.
This method likely stains by two mechanisms: ionic attachment of iron mordanted hematoxylin to most structures and by van der Waal’s forces to elastic fibres. The differentiation step is accomplished either by oxidative bleaching of hematoxylin by ferric chloride, or by displacement of the mordanted hematoxylin by free mordant in solution. The dye attached to elastic fibres by van der Waal’s forces is not extracted easily. It is, however, still susceptible to bleaching, which likely explains why fine fibres may be pale. That bleaching takes place is shown by the differentiating fluid remaining clear during differentiation, extracted dye being seen to bleach as it dissolves out.
Note that nuclei are only adequately, not well, stained.

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
Culling, C.F.A., (1963)
Handbook of histopathological techniques, 2nd ed.
Butterworths, London.

March 30, 2023

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Garvey’s Stain for Elastic, Fibrin & Collagen

By Carmen WongDye Type, Elastic Fibers, Fibrin, Iron Hematoxylin, Protocols, Stain Target, Stain Type

Protocol

Garvey's Stain

for Elastic, Fibrin & Collagen

steps

materials

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Materials

Stock Verhoeff A

Material	Amount
Hematoxylin	3	g
Ethanol, absolute	100	mL

Stock Verhoeff B

Material	Amount
Ferric chloride	2.3	g
Distilled water	100	mL

Stock Verhoeff C

Material	Amount
Potassium iodide	4	g
Iodine	2	g
Distilled water	100	mL

Working Verhoeff

Material	Amount
Stock solution A	3	volumes
Stock solution B	2	volumes
Stock solution C	1	volume

Polyacid

Material	Amount
Phosphotungstic acid	2	g
Distilled water	100	mL

Acetic water

Material	Amount
Acetic acid, glacial	0.5	mL
Distilled water	100	mL

Picro-mercuric chloride

Material	Amount
Picric acid, sat. aqu.	100	mL
Mercuric chloride	5	g
Distilled water	100	mL

Erythrocyte stain

Material	Amount
Lissamine fast yellow	1	g
Acetic acid, glacial	0.5	mL
Distilled water	100	mL

Plasma stain

Material	Amount
Biebrich scarlet	0.75	g
Acid fuchsin	0.75	g
Ponceau 2R	0.75	g
Acetic acid, glacial	0.5	mL
Distilled water	100	mL

Material	Amount for Var I		Amount for Var II
Aniline blue	0.5	g	–
Light green SF yellowish	–		2	g
Acetic acid, glacial	1	mL	2	mL
Distilled water	100	mL	100	mL

Tissue Sample

Formol sublimate fixation is preferred, although 10% formalin variants are acceptable. Paraffin sections at 3µ are preferred.

Protocol

Bring sections to water via xylene and ethanol.
1. Optionally, place into picro-mercuric chloride for 1 hour.
2. Wash well with warm water to remove picric acid.
Place in verhoeff’s solution for 9 minutes.
Wash with warm tap water for 5 minutes.
Place in lissamine fast yellow for 2 minutes.
Rinse with 0.5% acetic acid.
Place in the plasma stain for 5 minutes.
Rinse with distilled water.
Place in polyacid for 10 minutes.
Rinse with distilled water.
Place in a fiber stain variant for 2 minutes.
Rinse with 0.5% acetic acid.
Dehydrate, clear and mount with a resinous medium.

Expected Results

Nuclei – black
Elastic fibres – black
Fibrin – young – yellow
Fibrin – mature – red
Fibrin – old – blue or green
Erythrocytes – yellow
Muscle – red
Collagen – blue or green (some may be red)

Notes

Sections should be treated with picro-mercuric chloride if formol sublimate or similar fixation was not used initially.
The ferric chloride should be the hexahydrate crystals.
Although the iodine content of the Verhoeff elastic stain component may remove any mercury pigment, in methods such as this, some technologists prefer to apply the iodine-thiosulphate sequence before staining.

Safety Note

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

References

Garvey W., et. al., (1987),
A combined elastic, fibrin and collagen stain
Stain Technology, V. 62, No 6, pp 365-367.

March 30, 2023

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Garvey-Movat Pentachrome for Elastic, Mucin & Collagen

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

Protocol

Garvey-Movat Pentachrome

for Elastic, Mucin & Collagen

steps

materials

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Expected Results

Movat staining showing amyloid, lipofuscin, and myocardial fibrosis in an autopsy of senile cardiac amyloidosis

fig1-movat-staining-amyloid-lipofuscin-myocardial-fibrosis-autopsy-senile-cardiac-amyloidosis-600x400

Figure 1. Movat Staining Showing Elastic Fibres in an Autopsy Specimen of Senile Cardiac Amyloidosis

Movat staining of an autopsy specimen of senile cardiac amyloidosis shows nuclei and elastic fibres (in black), collagen and reticular fibers (in yellow), ground substance and mucin (in blue), fibrin (in bright red), and muscle (in red). Amyloid is visible as extracellular muddy brown material (right of image), abundant lipofuscin as dark red granular material, and myocardial fibrosis as yellow (left of image). Cardiac amyloidosis very high mag movat by Nephron / Michael Bonert, Pathology & Molecular Medicine, McMaster University is licensed under CC BY-SA 3.0.

Nuclei – black
Elastic fibres – black
Fibrin – mature – red
Muscle – red
Collagen – yellow
Ground substance – blue-green

Materials

Stock Verhoeff A

Material	Amount
Hematoxylin	3	g
Ethanol, absolute	100	mL

Stock Verhoeff B

Material	Amount
Ferric chloride	2.3	g
Distilled water	100	mL

Stock Verhoeff C

Material	Amount
Potassium iodide	4	g
Iodine	2	g
Distilled water	100	mL

Working Verhoeff

Material	Amount
Stock solution A	3	volumes
Stock solution B	2	volumes
Stock solution C	1	volume

Polyacid

Material	Amount
Phosphotungstic acid	2	g
Distilled water	100	mL

Acetic water

Material	Amount
Acetic acid, glacial	0.5	mL
Distilled water	100	mL

Picro-mercuric chloride

Material	Amount
Picric acid, sat. aqu.	100	mL
Mercuric chloride	5	g
Distilled water	100	mL

Alcian blue

Material	Amount
Alcian blue	1	g
Acetic acid, glacial	3	mL
Distilled water	100	mL

Plasma stain

Material	Amount
Biebrich scarlet, 1% aqu.	8	mL
Acid fuchsin, 1% aqu.	2	mL
Acetic acid, 1% aqu.	100	mL

Fibre stain

Material	Amount
Saffron du Gatinais	6	g
Ethanol, absolute	100	mL

Preparation

Add the saffron to the ethanol and seal the container.
Incubate at 56°C for 2 weeks. This solution must be anhydrous.

Tissue Sample

Formol sublimate fixation is preferred, although 10% formalin variants are acceptable. Paraffin sections at 3µ are preferred.

Protocol

Bring sections to water via xylene and ethanol.
1. Optionally, place into picro-mercuric chloride for 1 hour.
2. Wash well with warm water to remove picric acid.
Rinse with 3% acetic acid.
Place in alcian blue at 60°C for 10 minutes
Rinse with distilled water.
Place in working Verhoeff’s solution for 6 minutes.
Wash with warm tap water for 6 minutes.
Place in the plasma stain for 3 minutes
Rinse with distilled water.
Place in the polyacid for 15 minutes.
Rinse with 1% acetic acid.
Dehydrate thoroughly with absolute ethanol 3 changes.
Place in the fiber stain for 5-6 minutes.
Dehydrate, clear and mount with a resinous medium

Notes

Sections should be treated with picro-mercuric chloride if formol sublimate or similar fixation was not used initially.
The ferric chloride should be the hexahydrate crystals.
Sections thicker than 4µ may need the elastic stain differentiated by treating with 1% aqueous ferric chloride for 20-30 seconds, then washing well with tap water.
Although the iodine content of the Verhoeff elastic stain component may remove any mercury pigment, in methods such as this, some technologists prefer to apply the iodine-thiosulphate sequence before staining.

Safety Note

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

References

Garvey W., et. al., (1986),
Improved Movat pentachrome stain
Stain Technology, V. 61, No 1, pp 60-62.

March 30, 2023

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Iron Hematoxylin