Csaba's Stain

for Mast Cells

steps

materials

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Materials

Staining solution

Material	Amount
Alcian blue	0.36	g
Safranin	0.18	g
Ferric ammonium sulfate	0.48	g
Walpole’s acetate HCl buffer	100	mL

Walpole’s acetate-HCl buffer, pH 1.42

Material	Amount
Sodium acetate, M/1	100	mL
Hydrochloric acid, M/1	120	mL

Tissue Sample

Paraffin sections of neutral buffered formalin fixed tissue are suitable. Mercuric chloride fixatives are reputed to emphasise metachromasia. Other fixatives may be satisfactory.

Protocol

Bring sections to water via xylene and ethanol.
Place in to staining solution for 10-20 minutes.
Rinse with water.
Dehydrate with tertiary butanol.
clear with xylene and mount using a resinous medium.

Expected Results

Young mast cells – blue
Mature mast cells – red

Notes

To make an M/1 solution of sodium acetate, dissolve 82.04 grams anhydrous or 136.09 crystal sodium acetate in one litre of distilled water.
To make an M/1 solution of hydrochloric acid, dilute concentrated hydrochloric acid, which is about 10M, with distilled water (one part acid to 8 parts water) and titrate against an alkali of known molarity. Adjust the concentration to M/1 with distilled water.

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. pp. 169 & 419
Butterworths, London, England.

March 30, 2023

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Dahlia-Iron-Resorcin for Elastic Fibres

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

Protocol

Dahlia-Iron-Resorcin

for Elastic Fibres

steps

materials

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Materials

- Weigert’s iron hematoxylin or equivalent
- Van Gieson’s picro-fuchsin
- Iron-resorcin-dahlia solution
  Material Amount
  Dahlia 2 g
  Resorcin 4 g
  Ferric nitrate, 30% aqu. 50 mL
  0.5% aqu. hydrochloric acid 200 mL
  Methanol 180 mL
  Acetone 20 mL
  Hydrochloric acid, conc. 4 mL

Material	Amount
Dahlia	2	g
Resorcin	4	g
Ferric nitrate, 30% aqu.	50	mL
0.5% aqu. hydrochloric acid	200	mL
Methanol	180	mL
Acetone	20	mL
Hydrochloric acid, conc.	4	mL

Preparation

Bring 200 mL of 0.5% hydrochloric acid in an oversize flask to the boiling point, remove from the heat and carefully add the dahlia.
Return to the boil, and add the resorcin and ferric nitrate.
Continue boiling for 3 minutes, then cool and filter. Discard the filtrate. Dry the filter paper and beaker, then place the precipitate and filter paper back into the flask.
Add the methanol, acetone and hydrochloric acid.
Heat carefully on a hot plate until the precipitate dissolves.

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.
Place into the staining solution for 30 minutes or longer.
Wash with 95% ethanol to remove excess solution.
Differentiate with 1% acid alcohol if necessary.
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

Between the 1st and 2nd editions of their book, Bancroft and Stevens changed the solvent for the precipitate to the following:
Solvent Amount
2-methoxyethanol 100 mL
Distilled water 100 mL
Hydrochloric acid, conc. 4 mL
Dry the filter paper and beaker, then place the precipitate and filter paper back into the flask. Add the mixture and heat carefully on a hot plate until the precipitate dissolves.
2-methoxyethanol is also known as ethylene glycol monomethyl ether.
The authors suggest the following dyes as suitable substitutes for dahlia:
- Methyl violet
- Ethyl violet
- Victoria blue
- Thionin
Note that this method uses ferric nitrate instead of ferric chloride.

Solvent	Amount
2-methoxyethanol	100	mL
Distilled water	100	mL
Hydrochloric acid, conc.	4	mL

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.,
Theory and Practice of Histological Techniques, Ed. 1 (1977),
Churchill Livingstone, London, UK.
Bancroft, J. D. and Stevens, A.,
Theory and Practice of Histological Techniques, Ed. 2 (1982),
Churchill Livingstone, London, UK.

March 30, 2023

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Feulgen Nucleal Reaction for DNA

By Carmen WongAldehydes, Nucleal Reaction, Protocols, Schiff's Reagent Reactions, Stain Target, Stain Type

Protocol

Feulgen Nucleal Reaction

for DNA

steps

materials

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Materials

Hydrochloric acid, 1N
Schiff’s reagent
Light green, 1% aqueous

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Many other fixatives are satisfactory. Fixatives containing strong acids should be avoided as this method depends on the acid hydrolysis of DNA, and acids in some fixatives may pre-hydrolyse the tissue (picric acid in Bouin’s aqueous formal-picric-acetic mixture, for example).

Protocol

Bring sections to water via xylene and ethanol.
Rinse briefly with cold 1N hydrochloric acid.
Place into prewarmed hydrochloric acid for the appropriate time at 60°C.
Rinse briefly with cold 1N hydrochloric acid.
Rinse briefly with distilled water.
Place into Schiff’s reagent for 30-60 minutes at room temperature.
Give three sulphite rinses of about 1 minute each.
Wash well with water.
Optionally, counterstain with light green for 1 minute.
Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

DNA – red
Background – green

Notes

The appropriate time in hydrochloric acid varies depending on the fixative. The times given below are in minutes, but should be considered a guide only. Trials should be conducted to determine the optimum.
Fixative Time
Bouin Do not use
Carnoy 8
Champy 25
Flemming 16
Formalin (NBF) 10
Formal sublimate 8
Helly 8
SuSa 18
Zenker 5
A modification of this reaction uses more concentrated hydrochloric acid at room temperature. At step 3, place into 5N hydrochloric acid at room temperature for the appropriate time. Then continue on with step 4. This variant is considered to produce darker staining and a smaller loss of DNA.
Fixative Time
Alcoholic fixatives 20 minutes to 2 hours
Formalin containing fixatives 35 minutes to 4 hours
Formalin vapour 2 to 8 hours
Sulphite rinses are now considered unnecessary. They were used originally in the belief that placing directly into water would recolour the Schiff’s reagent and give false positive staining. It is now known that this is not so, providing the Schiff’s reagent is completely washed off. Sulphite rinses consist of:
Material Amount
Potassium metabisulphite, 10% aqu. 5 mL
Hydrochloric acid, 1N 95 mL

Fixative	Time
Bouin	Do not use
Carnoy	8
Champy	25
Flemming	16
Formalin (NBF)	10
Formal sublimate	8
Helly	8
SuSa	18
Zenker	5

Fixative	Time
Alcoholic fixatives	20 minutes to 2 hours
Formalin containing fixatives	35 minutes to 4 hours
Formalin vapour	2 to 8 hours

Material	Amount
Potassium metabisulphite, 10% aqu.	5	mL
Hydrochloric acid, 1N	95	mL

Safety Note

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

References

Pearse, A. G. E., (1968, 1972)
Histochemistry: Theoretical and Applied, Ed. 3
Churchill Livingstone, Edinburgh, London, UK

March 30, 2023

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Trevan and Sharrock’s Methyl Green – Pyronin

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

Protocol

Trevan and Sharrock's

Methyl Green – Pyronin

steps

materials

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Materials

Methyl green stock

Material	Amount
Methyl green	2	g
Distilled water	100	mL

Purifying the methyl green

Before making the methyl green-pyronin stock solution the methyl green must be purified to remove any methyl violet which has formed.

Place the 2% aqueous stock methyl green solution in a stoppered separating funnel.
Add some chloroform and shake periodically for about a half hour.
Crystal violet will discolor the chloroform as it is removed.
Allow to separate, then drain off the methyl green and discard the chloroform.
Repeat the extraction until the chloroform is colorless or almost so.

Pyronin Y stock

Material	Amount
Pyronin Y	5	g
Distilled water	100	mL

Acetate buffer pH4.8

Material	Amount
0.1M sodium acetate	119	mL
0.1M acetic acid	81	mL

Methyl green–pyronin stock

Material	Amount
Methyl green, 2% aqueous, purified	10	mL
Pyronin Y, 5% aqueous	17.5	mL
Distilled water	250	mL

Staining solution

Material	Amount
Methyl green–pyronin stock	1	volume
Acetate buffer pH 4.8	1	volume

Make fresh.

Tissue Sample

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

Protocol

Bring sections to water via xylene and ethanol.
Rinse with distilled water, then gently blot dry.
Place into the staining solution for 20–30 minutes,
Rinse with distilled water, then gently blot dry.
Dehydrate rapidly with acetone.
Clear with xylene and mount with a resinous medium.

Expected Results

DNA – blue–green
RNA – Red

Notes

Mercuric chloride fixation may cause the DNA to depolymerize during fixation and should be avoided. The differential staining depends on differences in the degree of polymerization of the two nucleic acids and depolymerization may cause the DNA to stain with pyronin.
Acid decalcified tissues are often unsatisfactory and may stain excessively red with pyronin. EDTA decalcification is usually satisfactory.
The extracted solutions of methyl green are reasonably stable, but changes in the expected nuclear coloration may require a freshly extracted solution of methyl green to be prepared.
This method is reasonably selective for nucleic acids but should be controlled with ribonuclease and/or deoxyribonuclease enzyme extractions if specificity is required.
Since this technique is quite simple, it is often recommended for plasma cells. Nissl substance also stains well.

Safety Note

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

References

Drury, R A, and Wallington, E A, (1967).
Carleton’s histological technique., Ed. 4, p. 164.
Oxford University Press, London, England.

March 30, 2023

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Pasini’s Trichrome for Elastic and Collagen

By Carmen WongDye Type, Elastic Fibers, Orcein, Protocols, Stain Target, Stain Type, Trichrome Staining, Trichrome, One-Step

Protocol

Pasini's Trichrome

for Elastic and Collagen

steps

materials

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Materials

Ehrlich’s alum hematoxylin
Solution A
Material Amount
Phosphotungstic acid 2 g
Distilled water 100 mL
Solution B – Var I
Material Amount
Eosin B 0.7 g
Acid fuchsin, sat. aqu. 4 mL
Unna’s elastic stain 35 mL
Glycerol 40 mL
Ethanol, 50% 35 mL
Solution B – Var II
Material Amount
Eosin B, 2% in 50% ethanol 30 mL
Acid fuchsin, sat. aqu. 4 mL
Unna’s elastic stain 30 mL
Glycerol 15 mL

Material	Amount
Eosin B	0.7	g
Acid fuchsin, sat. aqu.	4	mL
Unna’s elastic stain	35	mL
Glycerol	40	mL
Ethanol, 50%	35	mL

Material	Amount
Eosin B, 2% in 50% ethanol	30	mL
Acid fuchsin, sat. aqu.	4	mL
Unna’s elastic stain	30	mL
Glycerol	15	mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

Protocol

Bring sections to water via xylene and ethanol.
Stain nuclei with an alum hematoxylin nuclear stain.
Place into solution A for 10 minute.
Rinse quickly with distilled water.
Place into solution B for 15-20 minutes.
Rinse quickly with 70% ethanol.
Dehydrate and differentiate with 100% ethanol for a few seconds.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Elastic fibres – purple
Erythrocytes – orange
Collagen – blue

Notes

Solution B var I was given by Gray, var II was given by Gatenby & Beams.

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
Bolles Lee, A.. Edited by Gatenby, J.B. and Beams, H.W., (1950)
The Microtomist’s Vade-Mecum. 11 ed., pp. 485
Churchill, London, UK.

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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Silverman-Movat Pentachrome for Elastic, Mucin & Collagen

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

Protocol

Silverman-Movat Pentachrome

for Elastic, Mucin & Collagen

steps

materials

print

Expected Results

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

Nuclei – dark purple to black
Elastic fibres – purple to black
Muscle – Red, with longitudinal myofibrils, cross striations and intercalated discs delineated
Collagen and bone – yellow to yellow green
Mucin – blue
Ground substance – blue green
Cytoplasm – pink to brownish to red

Materials

Stock solution A

Material	Amount
Orcein	1	g
Hydrochloric acid, conc.	1	mL
Ethanol, 70%	500	mL

Stock solution B

Material	Amount
Hematoxylin	8	g
Ethanol, absolute	160	mL

Stock solution C

Material	Amount
Ferric chloride	9.6	g
Distilled water	90	mL

Stock solution D

Material	Amount
Iodine	1	g
Potassium iodide	2	g
Distilled water	97	mL

Working elastic solution

Material	Amount
Stock solution A	25	mL
Stock solution B	8	mL
Stock solution C	5	mL
Stock solution D	5	mL

Differentiator

Material	Amount
Stock C	10	mL
Distilled water	40	mL

Alcian blue

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

Ammoniated ethanol

Material	Amount
Strong ammonia	5	mL
Ethanol, 95%	95	mL

Plasma stain

Material	Amount
Woodstain scarlet 1% aqu.	4	mL
Acid fuchsin 1% aqu.	1	mL
Acetic acid, 0.5%	95	mL

Polyacid

Material	Amount
Phosphotungstic acid	5	g
Distilled water	100	mL

Fiber stain

Material	Amount
Spanish saffron	6	g
Ethanol, absolute	96	mL

Preparation

Incubate together in a sealed container at 56°C for 48 hours. Cool.

Tissue Sample

Paraffin sections at 4-6µ are suitable. Bouin’s fixation is preferred. 10% neutral buffered formalin is satisfactory. If formalin is used, refix sections with Bouin’s fluid for one hour at 56°C, then wash in running tap water to remove the yellow colour.

Protocol

Bring sections to water via xylene and ethanol.
Stain in alcian blue for 20 min.
Rinse in distilled water.
Place in ammoniated ethanol for 10 min at 56°C.
Wash in running tap water for 2 min.
Rinse in distilled water.
Stain in working elastic solution for 2 hours.
Wash in running water until collagen is clear and elastic prominent.
Rinse in distilled water.
Place in the plasma stain for 3 minutes.
Place in 0.5% aqueous acetic acid for 30 seconds.
Differentiate in the polyacid until collagen is clear and ground substance is blue.
Rinse in 0.5% aqueous acetic acid for 30 seconds.
Place in three changes of absolute ethanol for 1 minute each.
Place in the fiber stain for 8 minutes.
Dehydrate quickly in absolute ethanol, 2 changes.
Clear in xylene, three changes.
Coverslip with a resinous mounting medium.

Notes

The working elastic solution should be used only once, then discarded.
If the elastic fibers are not clearly delineated at step 8 and the background is not clean, place in the differentiator for a few minutes, then wash well in tap water until they are sharp.
Differentiation with the polyacid at step 12 takes from 3-10 minutes.
The fiber stain (alcoholic saffron) may be used repeatedly until staining intensity decreases. It is important that this solution not be contaminated with water. Place some Drierite into it to keep it anhydrous.

Safety Note

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

References

Silverman, J., (1972)
Histologic v 2, N° 2.

March 30, 2023

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Double Oxidation Schiff

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

Protocol

Double Oxidation Schiff

steps

materials

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Double oxidation may be used with any procedure which demonstrates aldehydes, including Schiff’s reagent and methenamine silver reduction. Its use is not necessarily confined to staining fungi and it may be found useful when demonstrating other structures and the background stains too darkly.

Materials

Periodic acid – 1% aqueous
Analine-acetic block
Material Amount
Acetic acid, glacial 5 mL
Aniline oil 45 mL

Material	Amount
Acetic acid, glacial	5	mL
Aniline oil	45	mL

Tissue Sample

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

Protocol

Bring sections to water via xylene and ethanol.
Oxidize with periodic acid for 10 minutes.
Rinse well with tap water.
Treat with aniline-acetic for 30 minutes.
Rinse well with tap water.
Re-oxidize with periodic acid for 20 minutes.
Rinse well with tap water.
Rinse with distilled water.
Place in Schiff’s reagent for 10-30 minutes.
Wash off with distilled water.
Wash well with tap water for about 10 minutes.
Counterstain with Mayer’s hemalum for 2 minutes.
Wash well with tap water until hemalum is blued.
Dehydrate with ethanol, clear with xylene and coverslip using a resinous medium.

Expected Results

Oxidizable carbohydrates – red
Fungi – red
Nuclei & background – blue

Notes

The time periodic acid is applied in the first oxidation determines the amount of background staining eliminated. If there is only a small amount of this material, then all of it may be blocked
The first oxidation will also oxidize some of the target material, and this staining will also be inhibited. If the first oxidation is applied for too long, the depth of staining of the target material may be affected. The 10 minutes specified is usually enough.
The time periodic acid is applied in the second oxidation determines the depth of staining for the target material. 20 minutes is usually adequate, but extending it may give darker staining. At some point, extending the time in periodic acid will cease to produce darker staining as all available carbohydrate will have been oxidized.

Safety Note

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

References

Personal observation, B. Llewellyn,

March 30, 2023

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Lillie’s Long PAS for 1-2-Glycols

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

Protocol

Lillie's Long PAS

for 1-2-Glycols

steps

materials

print

The periodic acid Schiff reaction (PAS) is used to demonstrate the presence of 1-2-glycols, and is consequently an important method in the histochemistry of carbohydrates and the histological demonstration of many structures.

Lillie referred to this as

“The Periodic acid Leucofuchsin Method in Relation to Various Modifying Procedures”.

He describes its purpose as being

“To indicate at what points and in what sequence various modifying procedures should be introduced”.

Materials

A periodic acid solution.
A strong Schiff’s reagent.
Sodium metabisulfite, 0.5% aqueous, optional.
Mayer’s hemalum, or Weigert’s iron hematoxylin.
Picric acid, saturated aqueous, optional.
Orange G, 2% aqueous, optional.
Methyl blue, 0.1% in saturated aqueous picric acid, optional.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory, although glutaraldehyde should be avoided.

Protocol

Dewax paraffin sections with xylene.
Remove xylene with two changes of absolute ethanol.

Optionally, do one or more of the following procedures in the order given.

Procedure	Comments
Acetylation	Acetylation blocks hydroxyl and amino groups. Cartilage and glycogen are the most resistant. Other carbohydrates are more easily affected.
Benzoylation	Similar to acetylation, this also blocks hydroxyl and amino groups. Again, cartilage and glycogen are the most resistant while other carbohydrates are more easily affected.
Deacetylation (saponification)	Deacetylation (also called saponification) reverses the effects of acetylation in some, if not all of the materials which were blocked. The reversal is progressive, that is, some substances recover their stainabilty before others.
Methylation	Extended methylation causes some otherwise oxidisable carbohydrates to become non-reactive. Primarily, however, it inhibits basophilia.
Aldehyde block	If applied before periodic acid oxidation, pre-existing aldehydes are inhibited from reacting and will be unstained. If applied after periodic acid oxidation, aldehydes produced by the periodic acid are inhibited from reacting and will be unstained. A second section which has not been blocked can be used to prove that oxidation formed the aldehydes.
Amylase digestion	Amylase (diastase) is used to remove glycogen. This is either to prove that a subtance is glycogen, or to remove it so that other PAS positive materials other than glycogen may be more clearly seen.
Protease digestion	Proteases may also be used to remove carbohydrate-protein complexes, although this is less common than amylase digestion because the enzymes also digest other tissue proteins.
Reducing rinses	This is an acidified solution of potassium iodide and sodium thiosulphate applied following periodic acid to remove any iodate or periodate remaining in the tissue.
Methanol, Pyridine & Solvent extraction	PAS positive carbohydrate-lipid complexes, or glycolipids, may be removed with these solvents. They may be used at ambient or elevated temperature.

Ensure sections are in water or an appropriate concentration of ethanol.
Oxidize for the appropriate time in the selected periodic acid solution.
Wash five minutes in running water, or 70% ethanol if using an alcoholic variant.
Optionally, do one of the following.
1. Hotchkiss’ reducing rinse, or
2. Aldehyde block
Place in Schiff’s reagent for 10 minutes, or longer if specially required.
Transfer to three successive sulfite rinses, 2 minutes each.
Wash in running tap water for 10 minutes.
Optionally, stain nuclei.
Optionally, stain cytoplasm.
Dehydrate and differentiate with two changes each of 95% and absolute ethanol.
Clear with xylene and coverslip using a resinous medium.

Expected Results

1-2-glycols – red
Nuclei – blue or black
Cytoplasm – yellow or unstained

Notes

A strong Schiff’s reagent is recommended, i.e. one that contains 0.5 or 1 gram of pararosanilin per 200 mL solution.
Sections should be celloidinised before deacetylation, and it may be advisable after the other options in step 3.
Experience shows that the reducing rinses may be omitted.
Mayer’s hemalum or Weigert’s iron hematoxylin are suitable nuclear stains.
Sat. picric acid, 2% orange G, or picro-methyl blue are suitable cytoplasmic stains.

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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