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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Sheridan’s Stain for Elastic Fibres

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

Protocol

Sheridan's Stain

for Elastic Fibres

steps

materials

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Materials

Weigert’s iron hematoxylin or equivalent
Van Gieson’s picro-fuchsin
Sheridan’s solution
Material Amount
Crystal violet 2 g
Dextrin 2 g
Resorcin 4 g
Ferric chloride, 30% aqu. 25 mL
Ethanol 95% 200 mL
Distilled water 200 mL
Hydrochloric acid, conc. 4 mL

Material	Amount
Crystal violet	2	g
Dextrin	2	g
Resorcin	4	g
Ferric chloride, 30% aqu.	25	mL
Ethanol 95%	200	mL
Distilled water	200	mL
Hydrochloric acid, conc.	4	mL

Preparation

Add the dye, dextrin and resorcin to the water in an oversize flask. Bring to the boil, and add the ferric chloride.
Boil for 5 minutes.
Cool and filter.
Dry the filter paper and beaker. Place the precipitate and filter paper back into the flask.
Add the ethanol and heat carefully until the precipitate dissolves.
Add the hydrochloric acid.
Restore to 200 mL with 95% ethanol.

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 Sheridan’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 – green
Collagen – red
Cytoplasm – yellow
Nuclei – blue

Notes

This is similar to Weigert’s iron resorcin fuchsin, but substitutes crystal violet and dextrin for basic fuchsin.
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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Taenzer-Unna Orcein for Elastic Fibres

By Carmen WongDye Type, Elastic Fibers, Orcein, Protocols, Stain Target, Stain Type

Protocol

Taenzer-Unna Orcein

for Elastic Fibres

steps

materials

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Materials

Orcein solution

Material	Amount
Orcein	1	g
Ethanol, 70%	100	mL
Hydrochloric acid, conc.	1	mL

Acid ethanol

Material	Amount
Ethanol, 70%	100	mL
Hydrochloric acid, conc.	1	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.
Rinse with 70% ethanol.
Place into orcein solution for an appropriate time.
Optionally, treat with acid ethanol until collagen is unstained (a few seconds).
Wash with water
Counterstain if wished.
Dehydrate with 95% and absolute ethanols.
Clear with xylene and mount with a resinous medium.

Expected Results

Elastic fibres – purplish brown
Other tisse – as counterstained

Notes

Gray specifies 0.8 grams orcein, most others specify 1 gram.
Gray specifies 50% ethanol, Culling 70% and Carleton 80%. Others have recommended concentrations ranging from 65% to 100% (Lillie).
Staining times specified include 6-12 hours (Gray), overnight (Culling) and 30 minutes to 2 hours (Carleton).
Elevating the temperature (37°C Carleton, 56°C Culling) decreases staining time.
Recommended counterstains are van Gieson, methylene blue, H&E.

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:
von Kahlden, C., and Laurent, O. (1896)
Technique microscopique.
Carré, Paris, France
Drury, R.A.B. and Wallington, E.A., (1980)
Carleton’s histological technique Ed. 5
Oxford University Press, Oxford, UK.
Culling C.F.A., (1974)
Handbook of histopathological and histochemical techniques Ed. 3
Butterworth, London, UK.
Mallory, F. B. & Wright, J.H., (1904)
Pathological technique, Ed.3
W. B. Saunders, Philadelphia, USA.
Lillie, R.D., (1954)
Histopathologic technique and practical histochemistry Ed.2
Blakiston, New York, USA.

March 30, 2023

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Unna’s Orcein-Aniline Blue for Elastic

By Carmen WongDye Type, Elastic Fibers, Orcein, Protocols, Stain Target, Stain Type

Protocol

Unna's Orcein-Aniline Blue

for Elastic

steps

materials

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Materials

A red nuclear stain
Solution A
Material Amount
Acetic acid, glacial 1 mL
Distilled water 1 L
Solution B
Material Amount
Aniline blue 0.5 g
Orcein 0.5 g
Acetic acid, glacial 2.5 mL
Ethanol, 100% 25 mL
Distilled water 50 mL
Glycerol 10 mL

Material	Amount
Aniline blue	0.5	g
Orcein	0.5	g
Acetic acid, glacial	2.5	mL
Ethanol, 100%	25	mL
Distilled water	50	mL
Glycerol	10	mL

Preparation

Dissolve the aniline blue in the water with heat.
Cool and filter.
Dissolve the orcein in the ethanol.
Add the acetic acid and glycerol.
Combine the solutions.

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.
Stain the nuclei with a red nuclear stain.
Place into solution A for a few minutes.
Place into solution B for 1-10 hours.
Differentiate with solution A until elastic fibres are clear.
Dehydrate with 95% and absolute ethanols.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – red
Elastic – reddish brown
Bone – reddish brown
Background – blue

Notes

Gatenby & Beams specified water blue rather than aniline blue
Unna’s method has been more commonly applied in conjunction with other methods, such as Pasini’s trichrome, with solution B (Unna’s solution) being incorporated into other staining solutions.

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:
Gatenby, J. B. & Cowdry, E. V., (1928)
Bolles Lee’s Microtomist’s Vade Mecum, pp. 280
Blakiston, Philadelphia

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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Weigert’s Stain for Elastic Fibres

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

Protocol

Weigert's Stain

for Elastic Fibres

steps

materials

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Materials

Weigert’s iron hematoxylin or equivalent
Van Gieson’s picro-fuchsin
Weigert’s solution
Material Amount
Basic fuchsin 2 g
Resorcin 4 g
Ferric chloride, 30% aqu. 25 mL
Ethanol 95% 200 mL
Distilled water 200 mL
Hydrochloric acid, conc. 4 mL

Material	Amount
Basic fuchsin	2	g
Resorcin	4	g
Ferric chloride, 30% aqu.	25	mL
Ethanol 95%	200	mL
Distilled water	200	mL
Hydrochloric acid, conc.	4	mL

Preparation

Add the dye and resorcin to the water in an oversize flask. Bring to the boil, and add the ferric chloride.
Boil for 5 minutes.
Cool and filter. Dry the filter paper and beaker. Place the precipitate and filter paper back into the flask.
Add the ethanol and heat carefully until the precipitate dissolves.
Add the hydrochloric acid.
Restore to 200 mL with 95% ethanol.

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 Weigert’s solution for 20 minutes to 1 hour.
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 – blue black
Collagen – red
Cytoplasm – yellow
Nuclei – blue

Notes

The procedure often benefits from a Mallory bleach.
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

Culling C.F.A., (1974)
Handbook of histopathological and histochemical techniques, Ed. 3
Butterworth, London, UK.
Drury, R.A.B. and Wallington, E.A., (1980)
Carleton’s histological technique, Ed. 5
Oxford University Press, Oxford, 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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Engel & Cunningham’s Trichrome for Muscle Fibres, Types I & II

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

Protocol

Engel & Cunningham's Trichrome

for Muscle Fibres, Types I & II

steps

materials

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Materials

Harris alum hematoxylin
Solution A
Material Amount
Chromotrope 2R 0.6 g
Fast green FCF 0.3 g
Phosphotungstic acid 0.6 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Adjust to pH 3.4 with 1N NaOH.
Solution B
Material Amount
Acetic acid, glacial 0.2 mL
Distilled water 100 mL

Material	Amount
Chromotrope 2R	0.6	g
Fast green FCF	0.3	g
Phosphotungstic acid	0.6	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

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

Tissue Sample

Unfixed frozen sections of liquid nitrogen quenched muscle. Sections should be 5-10µ in thickness. The fibres should be in cross section, as close to a right angle to their length as possible.

Protocol

Cut cryostat sections and dry at room temperature.
Stain nuclei with Harris’ hematoxylin for 5 minutes.
Rinse well with distilled water.
Place into solution A for 10 minute.
Rinse with solution B for a few seconds to differentiate.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Type I fibres – green with a red cast
Type II fibres – green

Notes

Both fibre types are green overall, but type I fibres contain more red staining granular material and have a red cast.
This is a modification of Gomori’s trichrome.

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.
Citing:
Engel & Cunningham, (1963)
Rapid examination of muscle tissue.
An improved trichrome method for fresh frozen biopsy specimens.
Neurology, vol.13, pp.921

March 30, 2023

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Schmorl’s Stain for Reducing Substances

By Carmen WongIntracytoplasmic Granules, Melanin & Enterochromaffin, Metal Impregnation, Metal Impregnation, Non-Silver, Protocols, Stain Target, Stain Type

Protocol

Schmorl's Stain

for Reducing Substances

steps

materials

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Materials

Nuclear fast red
Schmorl’s solution
Material Amount
Ferric chloride, 1% aqueous, fresh 30 mL
Potassium ferricyanide, 1% aqueous, fresh 4 mL
Distilled water 6 mL

Material	Amount
Ferric chloride, 1% aqueous, fresh	30	mL
Potassium ferricyanide, 1% aqueous, fresh	4	mL
Distilled water	6	mL

This solution should be freshly prepared immediately before use.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Protocol

Bring sections to distilled water via xylene and ethanol.
Place into freshly made Schmorl’s solution for 10 minutes.
Wash well with water.
Counterstain with nuclear fast red.
Rinse well with water.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Reducing substances – blue
Nuclei – red

Notes

Reducing substances which may be present are colored blue. This includes melanin, for which it is a useful method, enterochromaffin and lipofuscin.
The Schmorl’s solution is from Lillie’s modification of Schmorl’s ferricyanide reduction method for tissue reducing substances.

Safety Note

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

References

Lillie, R.D., (1954)
Histopathologic technique and practical histochemistry Ed.2
Blakiston, New York, USA.

March 30, 2023

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Leder Esterase for Mast Cells

By Carmen WongIntracytoplasmic Granules, Mast Cells, Protocols, Stain Target

Protocol

Leder Esterase

for Mast Cells

steps

materials

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Materials

Pararosanilin

Material	Amount
Pararosanilin	0.5	g
Distilled water	20	mL
Hydrochloric acid, conc.	2.5	mL

Warm gently and filter. Refrigerate.

Nitrosylated pararosanilin

Material	Amount
Pararosanilin solution	0.1	mL
Sodium nitrite, 4% aqueous	0.1	mL

The sodium nitrite solution must be fresh. After mixing, let stand for one minute. Use immediately.

Sorenson Stock A

Material	Amount
Disodium hydrogen phosphate	2.73	g
Distilled water	250	mL

Sorenson Stock B

Material	Amount
Potassium dihydrogen phosphate	2.27	g
Distilled water	250	mL

Sorenson working buffer

Material	Amount
Sorenson stock A	41	mL
Sorenson stock B	9	mL

Incubating medium

Material	Amount
Naphthol-ASD chloroacetate	10	mg
N,N-dimethyformamide	1	mL
Sorenson’s working buffer	35	mL
Nitrosylated pararosanilin	0.2	mL

Combine in the order given. Mix well, filter and use immediately.

Light green

Material	Amount
Light green SF yellowish	1	g
Distilled water	100	mL
Glacial acetic acid	1	mL

Tissue Sample

4µ paraffin sections of formalin fixed tissue are suitable.

Protocol

Bring sections to distilled water.
Place in the incubating medium for 40 minutes.
Wash in running tap water for 5 minutes.
Counterstain with light green for 30 seconds.
Wash in running tap water for 5 min.
Air dry.
Clear in xylol and mount.

Expected Results

Esterase activity (mast cells) – red
Background – green

Notes

An alternative to air drying, which avoids some of the artifact introduced, is to blot the section, then wash with xylene. This is repeated until the sections are cleared.

Safety Note

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

References

Leder, L.D. (1964)
Klinische Wochenschrift

March 30, 2023

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