Herovici's Stain

for Young and Mature Collagen

steps

materials

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Materials

Weigert’s iron hematoxylin or the Celestine blue hemalum sequence
Solution A
Material Amount
Picric acid, sat. aqu. 100 mL
Acid fuchsin, 1% aqu. 10 mL
Staining solution
Material Amount
Solution A 50 mL
Methyl blue, 0.05% aqu. 50 mL
Glycerol 10 mL
Lithium carbonate, sat. aqu. 0.5 mL

Material	Amount
Picric acid, sat. aqu.	100	mL
Acid fuchsin, 1% aqu.	10	mL

Material	Amount
Solution A	50	mL
Methyl blue, 0.05% aqu.	50	mL
Glycerol	10	mL
Lithium carbonate, sat. aqu.	0.5	mL

Tissue Sample

Paraffin sections at 5µ of formol-acetic-ethanol (10:5:85) fixed tissue was recommended.

Protocol

Bring sections to water via xylene and ethanol.
Stain nuclei with the acid resistant nuclear stain.
Rinse well with tap water.
Place into the staining solution for 2 minutes.
Wash with 1% acetic acid for 2 minutes.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Young collagen and reticulin – blue
Mature collagen – red
Cytoplasm – yellow
Nuclei – black

Notes

Solution A is van Gieson’s solution.
Cook notes that the original included a final step with metanil yellow for cytoplasmic staining, which he omitted.

Safety Note

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

References

Cook, H. C., (1974)
Manual of Histological Demonstration Techniques
Butterworths, London, UK.
Citing:
Herovici, c., (1963)
Stain Technology, v. 38, p. 204

March 30, 2023

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Puchtler’s Picro-Sirius red for Collagen

By Carmen WongDye Type, Picro-Fuchsin, Protocols, Stain Type, Trichrome Staining

Protocol

Puchtler's Picro-Sirius red

for Collagen

steps

materials

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Materials

Picro-sirius red

Material	Amount
Sirius red F3B	0.5	g
Saturated aqueous picric acid	500	mL

Acetic acid water

Material	Amount
Acetic acid, glacial	5	mL
Distilled water	11	mL

Tissue Sample

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

Protocol

Bring sections to water.
Optionally, stain nuclei with an acid resistant nuclear stain.
Wash well with water.
Place into Picro-sirius red solution for 1 hour.
Wash with two changes of acetic acid water.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Light Microscopy

Nuclei – black if stained
Collagen – red
Cytoplasm – yellow

Polarising Microscopy

Large fibres – yellow or orange birefringence
Thin fibres – green birefringence

Notes

This method can be used as an alternative for van Gieson’s stain or, using polarising microscopy, as a sensitive method for collagen.

Safety Note

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

References

Kiernan. J.A., (1999)
Histological and histochemical methods: Theory and practice, Ed. 3
Butterworth Heinemann, Oxford, UK.

March 30, 2023

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van Gieson’s Stain for Collagen

By Carmen WongDye Type, Picro-Fuchsin, Protocols, Stain Type, Trichrome Staining

Protocol

van Gieson's Stain

for Collagen

steps

materials

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Materials

Acid resistant nuclear stain: Weigert’s iron hematoxylin or Celestine blue hemalum
Staining solution
Material Amount
Saturated aqueous Picric acid 100 mL
1% aqueous Acid fuchsin 5-15 mL

Material	Amount
Saturated aqueous Picric acid	100	mL
1% aqueous Acid fuchsin	5-15	mL

Tissue Sample

Paraffin sections at 5µ are suitable. Many fixatives, including formalin, are satisfactory. Stains using acid dyes often benefit from picric acid or mercuric chloride fixation.

Protocol

Bring sections to water via xylene and ethanol.
Stain nuclei with the acid resistant nuclear stain.
Rinse well with tap water.
Place into the staining solution for 2-5 minutes.
Optionally, rinse quickly with distilled water.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Collagen – red
Cytoplasm – yellow
Nuclei – black

Notes

This method is often used to counterstain other primary staining methods. In that case the nuclear stain may not be necessary and should be ommitted.

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.
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.

March 30, 2023

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Kohashi’s Trichrome for Elastic and Collagen

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

Protocol

Kohashi's Trichrome

for Elastic and Collagen

steps

materials

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Materials

Solution A

Material	Amount
Azocarmine	0.1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Solution B

Material	Amount
Aniline	0.1	mL
Ethanol, 95%	100	mL

Solution C

Material	Amount
Acetic acid, glacial	1	mL
Ethanol, 95%	100	mL

Solution D

Material	Amount
Phosphotungstic acid	5	g
Distilled water	100	mL

Solution E

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

Tissue Sample

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

Protocol

Bring sections to water via xylene and ethanol.
Place into solution A for 12-15 minutes.
Rinse quickly with distilled water.
Differentiate nuclei with solution B.
Place into solution C for 30-60 seconds.
Rinse with distilled water.
Place into solution D for 30-60 minutes.
Rinse with distilled water.
Place into solution E for 15-20 minutes.
Place into 95% ethanol until differentiated.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

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

Notes

Solution E is Pasini’s trichrome solution.

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:
Kohashi, (1937)
Folia anatomica Japonica, v.15, pp.175

March 30, 2023

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Mollier’s trichrome for Elastic and Collagen

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

Protocol

Mollier's trichrome

for Elastic and Collagen

steps

materials

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Materials

Weigert’s iron hematoxylin or equivalent
Solution A
Material Amount
Orcein 0.8 g
Hydrochloric acid 1 mL
Ethanol, 100% 50 mL
Distilled water 50 mL
Solution B
Material Amount
Azocarmine 2 g
Acetic acid, glacial 1 mL
Distilled water 100 mL
Solution C
Material Amount
Phosphotungstic acid 5 g
Distilled water 100 mL
Solution D
Material Amount
Naphthol green B 1 g
Acetic acid, glacial 1 mL
Distilled water 100 mL

Material	Amount
Orcein	0.8	g
Hydrochloric acid	1	mL
Ethanol, 100%	50	mL
Distilled water	50	mL

Material	Amount
Azocarmine	2	g
Acetic acid, glacial	1	mL
Distilled water	100	mL

Material	Amount
Naphthol green B	1	g
Acetic acid, glacial	1	mL
Distilled water	100	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.
Place into solution A for 12 hours.
Stain nuclei with Weigert’s iron hematoxylin for 1-3 minutes.
Differentiate the nuclear stain if necessary.
Wash with water for 15 minutes.
Place into solution B for 15-30 minutes.
Rinse with distilled water.
decolorise in solution C for 2-6 hours, changing the solution three times.
Rinse quickly with distilled water.
Place into solution D for 15-30 minutes.
Agitate vigorously in 95% ethanol for 30 seconds.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Elastic – black
Erythrocytes – red
Cytoplasm – purple
Collagen – green

Notes

Solution A is the Unna-Taenzer elastic solution.

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:
Mollier, (1938)
Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik, v.55, pp.472.
And:
von Kahlden, C. and Laurent, O., (1896)
Technique microscopique, pp.143
Carré, Paris, France

March 30, 2023

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Bauer Reaction for Carbohydrates

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

Protocol

Bauer Reaction

for Carbohydrates

steps

materials

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Materials

A Schiff reagent
A progressive hemalum, such as Mayer
Chromic acid
Material Amount
Chromium trioxide 4 g
Distilled water 100 mL
Sulfurous acid
Material Amount
Sodium metabisulfite, 10% aqu. 6 mL
Hydrochloric acid, 1N 5 mL
Distilled water 100 mL

Material	Amount
Chromium trioxide	4	g
Distilled water	100	mL

Material	Amount
Sodium metabisulfite, 10% aqu.	6	mL
Hydrochloric acid, 1N	5	mL
Distilled water	100	mL

Tissue Sample

Protocol

Bring sections to water via xylene and ethanol.
Oxidize in chromic acid for 40-60 minutes.
Rinse with tap, then distilled water.
Place into Schiff’s reagent for 15 minutes.
Place into sulfurous acid rinses, 3 changes of 2 minutes each.
Wash with running tap water.
Counterstain with hemalum for 1 minute, and blue
Dehydrate with ethanols.
Clear with xylene and mount with a resinous medium.

Expected Results

Glycogen, mucin – red
Fungi – red
Nuclei – blue

Notes

Modern practice is to leave out the sulfite rinses and wash with large amounts of tap water.
A progressive hemalum should be used as counterstain because regressive hemalums sometimes stain mucin.
Mucins are not usually as dark as with a PAS.
Applying chromic acid for too long weakens staining due to continued oxidation of the aldehydes first produced.

Safety Note

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

References

McManus, J. F. A. and Mowry, R. W., (1960)
Staining Methods Histologic and Histochemical
Harper & Row, New York, NY, USA.

March 30, 2023

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Bennett’s Alum Hematoxylin

By Carmen WongDye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type

Protocol

Bennett's Alum Hematoxylin

steps

materials

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Materials

Material	Amount	Function
Hematoxylin	1 g	Dye
Potassium alum	90 g	Mordant
Distilled water	1 L	Solvent
Sodium iodate	0.2 g	Oxidant
Chloral hydrate	50 g	Stabiliser
Citric acid	1 g	Acidifier

Compounding procedure

Heat the water. Then add in order:
1. Hematoxylin
2. Sodium iodate
3. Alum
4. Citric acid
5. Chloral hydrate.
Note: Dissolve each ingredient before adding the next.
Cool to room temperature and filter. It may be used immediately.

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for 5-20 minutes.
Rinse with water and blue.
Rinse well with water.
Counterstain if desired.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Background – as counterstain or unstained

Notes

Putt gives no information regarding staining time and characteristics, but the similarity of the formula to Mayer’s (Langeron’s) hemalum suggests it is progressive and selectively nuclear. A staining time of 10-20 minutes should be adequate.
Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

Safety Note

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

References

F. A. Putt
Manual of Histopathological Staining Methods
John Wiley & Sons, New York, NY., USA

March 30, 2023

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Bencosme’s Alum Hematein

By Carmen WongDye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type

Protocol

Bencosme's Alum Hematein

steps

materials

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Materials

Original

Material	Amount	Function
Hematein	2 g	Dye
Potassium alum	133 g	Mordant
Distilled water	920 mL	Solvent
Glacial acetic acid	20 mL	Acidifier

Variant

Material	Amount	Function
Hematein	2.5 g	Dye
Potassium alum	120 g	Mordant
Distilled water	1 L	Solvent
Glacial acetic acid	10 mL	Acidifier

Compounding procedure

Add the alum to the water in a 2 L flask and bring to a boil for 1 minute.
Add the hematein and mix by swirling.
Place an inverted funnel over the flask and simmer for 10 minutes, shaking frequently.
Cool to room temperature and add the acetic acid.
Filter before use and twice weekly.
It may be used immediately.

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for 2 minutes.
Rinse with water.
Differentiate with 0.5% hydrochloric acid in 70% ethanol for one second.
Rinse well with water and blue.
Rinse well with water.
Counterstain with HPS.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue
Background – shades of red and yellow.

Notes

Note that this solution uses hematein rather than hematoxylin. Hematoxylin could be used, but would require 0.4 grams sodium iodate to be added along with the dye for complete oxidation in the original solution, although 0.3 grams might be more appropriate to extend the life. Comparable amounts would be 0.5 g and 0.4 g, respectively, for the variant formula.
When freshly made this solution stains in 2 minutes. Staining time slowly increases to 10 minutes as the solution ages. Life is about a month.
Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

Safety Note

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

References

Medical Laboratory Technology, 2nd ed. (1969)
Lynch M.J., Raphael S.S., Mellor L.D., Spare P.D. and Inwood M.J.H.,
W. B. Saunders Co., Toronto, On., Canada

March 30, 2023

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Bensley’s Copper Hematoxylin

By Carmen WongDye Type, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Type

Protocol

Bensley's Copper Hematoxylin

steps

materials

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Materials

Solution A

Material	Amount	Function
Hematoxylin,	1 g	Dye
Distilled water	100 mL	Solvent

Solution B

Material	Amount	Function
Cupric acetate,	to saturation	Mordant
Distilled water	100 mL	Solvent

Solution C

Material	Amount	Function
Potassium chromate,	5 g	–
Distilled water	100 mL	Solvent

Solution D

Material	Amount	Function
Weigert’s borax-ferricyanide	20 mL	Differentiator
Distilled water	80 mL	Diluent

Compounding procedure

Make each solution separately.

Protocol

Bring sections to water with xylene and ethanol.
Place into solution A for 5 minutes.
Rinse with water.
Dip into solution B.
Rinse with water.
Place into solution C for 2 minutes.
Return to solution A for 1 minute.
Place in solution D until differentiated.
Rinse well with water.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Nuclei and other structures – blue-black

Notes

After step 7, the sections should be intense black. Repeat steps 5-7 if necessary.
Bensley recommended prestaining with Mayers mucicarmine.
Best results are obtained with freshly made solutions

Safety Note

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

References

Bensley R. R. and Bensley, S. H., (1938)
Handbook of Histological and Cytological Technique.
U. Chicago Press, Chicago, USA

March 30, 2023

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Bensley’s trichrome for Muscle and Collagen

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

Protocol

Bensley's trichrome

for Muscle and Collagen

steps

materials

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Materials

Solution A

Material	Amount
Acid fuchsin	To saturation
Aniline water	100	mL

Solution Preparation

Add about 20 g dye to the aniline water.
Shake intermittently for 48 hours. Filter.

Solution B

Material	Amount
Phosphomolybdic acid	1	g
Distilled water	100	mL

Solution C

Material	Amount
Orange G	2	g
Aniline blue	0.5	g
Distilled water	100	mL

Tissue Sample

Protocol

Bring sections to water via xylene and ethanol.
Place into solution A for 10 minute.
Rinse with distilled water.
Place into solution B for 10 minutes.
Rinse quickly with distilled water.
Place into solution C for 60 minutes.
Rinse with 95% ethanol until clouds of dye cease being extracted.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – red
Erythrocytes – orange
Cytoplasm – red
Collagen – blue

Notes

Solution A is Altmann’s acid fuchsin, originally formulated for staining mitochondria.
This method does not specify an acid resistant nuclear stain. Doing so before staining with solution A may improve the technique.

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:
Mallory, F. B., (1938),
Pathological technique.
Saunders, Philadelphia, Pa, USA

March 30, 2023

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