Sweat and Puchtler's Sirius Red

for Amyloid

steps

materials

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Materials

Mayer’s hemalum
Neutral buffered formalin (pH 7.0)
0.1M borate or borate-phosphate buffer pH 9.0
Alkaline alcohol
Material Amount
Ethanol, 80% 100 mL
Sodium hydroxide, 1% aqueous 1 mL
Sirius red
Material Amount
Sirius red 1 g
Distilled water 100 mL
Sodium chloride 0.5 g

Material	Amount
Ethanol, 80%	100	mL
Sodium hydroxide, 1% aqueous	1	mL

Material	Amount
Sirius red	1	g
Distilled water	100	mL
Sodium chloride	0.5	g

Tissue Sample

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

Protocol

Bring sections to water via xylene and ethanol.
Place into neutral buffered formalin overnight.
Wash in tap water for 15 minutes.
Place into alkaline alcohol for 20 – 60 minutes.
Rinse well with distilled water.
Place into sirius red at 60°C for 60 – 90 minutes.
Rinse with buffer.
Wash with tap water for 5 minutes.
Stain nuclei with hemalum and blue.
Dehydrate with absolute ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Amyloid – red
Nuclei – blue
Background – colorless

Notes

This method uses sirius red F3B. The dye sirius red 4B is not suitable.
Sirius scarlet GG, CI 40270, may also be used.
Amyloid displays deep green birefringence when viewed with crossed polarizers, one above and one below the section.

Safety Note

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

References

Sweat, F. and Puchtler, H., (1965),
Demonstration of amyloid with direct dyes,
Archives of Pathology, v 80, page 613

March 30, 2023

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Thioflavine S For Senile Plaques & Neurofibrillary Tangles In Alzheimer’s

By Carmen WongAmyloid, Fluorescent Staining, Protocols, Stain Target, Stain Type

Protocol

Thioflavine S

For Senile Plaques & Neurofibrillary Tangles In Alzheimer's

steps

materials

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Materials

Potassium permanganate, 0.25% in distilled water
Ethanol, 70%
Acetic acid, 0.25%
Bleach solution
Material Amount
Potassium metabisulphite 1 g
Oxalic acid 1 g
Distilled water 100 mL
Blocking solution
Material Amount
Sodium hydroxide 1 g
Hydrogen peroxide, 30% 3 mL
Distilled water 100 mL
Thioflavine S
Material Amount
Thioflavine S 0.0125 g
Ethanol, 50% 100 mL
Glycerine Water
Material Amount
Glycerine 3 vols.
Distilled water 1 vol.

Material	Amount
Potassium metabisulphite	1	g
Oxalic acid	1	g
Distilled water	100	mL

Material	Amount
Sodium hydroxide	1	g
Hydrogen peroxide, 30%	3	mL
Distilled water	100	mL

Material	Amount
Thioflavine S	0.0125	g
Ethanol, 50%	100	mL

Material	Amount
Glycerine	3	vols.
Distilled water	1	vol.

Tissue Sample

30µ free floating sections of neutral buffered formalin fixed tissue are suitable. If paraffin embedded, they should be carefully dewaxed and hydrated before staining, but should remain free floating.

Protocol

Place in potassium permanganate for 20 minutes.
Rinse well with distilled water.
Place in the bleach solution for 2 minutes.
Rinse well with distilled water.
Place in the blocking solution for 20 minutes.
Rinse well with distilled water.
Place in acetic acid solution for 5 seconds.
Rinse well with distilled water.
Mount sections on microscope slides using an adhesive, dry, then rehydrate.
Place into thioflavine S solution for 3-5 minutes.
Rinse twice with 50% ethanol.
Rinse twice with distilled water.
Mount in glycerine water or glycerine jelly.

Expected Results

With appropriate filters, amyloid fluoresces bright yellow.

Notes

Although the method specifies an aqueous mounting medium, either blotting and treating with xylene repeatedly until clear, or dehydrating with ethanol and clearing with xylene, then mounting with a fluorescence free resinous medium may be satisfactory.

Safety Note

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

References

Guntern, R., Bouras, C., Hof, P.R. & Vallett, P.G., (1992),
An Improved Thioflavine S Method For Staining Neurofibrillary Tangles And Senile Plaques In Alzheimer’s Disease.

March 30, 2023

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Vassar & Culling’s Thioflavine T for Amyloid Fluorescence

By Carmen WongAmyloid, Fluorescent Staining, Protocols, Stain Target, Stain Type

Protocol

Vassar & Culling's Thioflavine T

for Amyloid Fluorescence

steps

materials

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Materials

Alum hematoxylin
Thioflavine T, 1% aqueous
Acetic acid, 1% aqueous

Tissue Sample

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

Protocol

Bring sections to water via xylene and ethanol.
Place into alum hematoxylin for 2 minutes.
Rinse well with water.
Place into thioflavine T solution for 3 minutes.
Rinse with water.
Place into acetic acid solution for 20 minutes.
Wash with water.
Mount in a fluorescence free aqueous mounting medium.

Expected Results

Using a UG1 or UG2 exciter filter and a UV barrier filter, or a BG12 exciter and an OG4 or OG5 barrier filter, amyloid fluoresces bright yellow.

Notes

The pretreatment with alum hematoxylin suppresses nuclear fluorescence.
Some workers have reported that materials other than amyloid may fluoresce yellow. The authors say this is caused by using a yellow barrier filter and strongly recommended the first filter combination. With this, these materials fluoresce white or pale blue.

Safety Note

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

References

Vassar, P.S. and Culling, C.F.A., (1959),
Fluorescent stains with special reference to amyloid and connective tissue,
Archives of pathology, v 68, page 487
Bancroft, J.D. and Stevens A. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.

March 30, 2023

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Wolman’s Standard Toluidine Blue (STB) for Amyloid

By Carmen WongAmyloid, Protocols, Stain Target

Protocol

Wolman's Standard Toluidine Blue(STB)

for Amyloid

steps

materials

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Materials

Material	Amount
Toluidine blue	1	g
Distilled water	50	mL
Iso-propanol, absolute	50	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 in the toluidine blue solution at 37°C for 30 minutes.
Blot carefully.
Place into absolute iso-propanol for one minute.
Blot carefully.
Clear with xylene and coverslip using Canada balsam.
Examine microscopically using crossed polarizing filters.

Expected Results

Amyloid – orange-red to red birefringence.
Orthochromatic tissue – blue-white birefringence
metachromatic tissue – yellow-green birefringence

Notes

Wolman strongly recommended this procedure, considering it to be highly selective for amyloid.
The birefringence is independent of section thickness and the quality of the microscope optics.

Safety Note

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

References

Wolman, M. (1971).
Amyloid, its nature and molecular structure: comparison of a new toluidine blue polarized light method with traditional procedures.
Laboratory Investigation, v. 25: p. 104-110.

March 30, 2023

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Birch-Hirschfeld’s Crystal Violet for Amyloid

By Carmen WongAmyloid, Metachromasia, Protocols, Stain Target, Stain Type

Protocol

Birch-Hirschfeld's Crystal Violet

for Amyloid

steps

materials

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Materials

Bismarck brown, saturated aqueous
Crystal violet, 0.5% aqueous
Acetic acid, 1% aqueous
Levulose syrup

Tissue Sample

Frozen sections are preferred. Cryostat sections usually show brighter metachromasia. Unmounted frozen sections may also be floated in each solution and mounted on a slide just before coverslipping. 5µ paraffin sections of neutral buffered formalin fixed tissue are likely also suitable. Other fixatives may be satisfactory.

Protocol

Bring sections to water via xylene and ethanol, except for cryostat and frozen sections.
Place into bismarck brown solution for 5 minutes.
Rinse well with 95% ethanol, then rinse with distilled water.
Place into crystal violet solution for 5 minutes.
Rinse with water.
If necessary, differentiate in 1% acetic acid until amyloid is red and contrasts well with the tissue.
Wash well in tap water.
Drain all water from the slide until just damp and mount with levulose syrup.
Ring the coverslip to inhibit evaporation of the mounting medium.

Expected Results

Amyloid – purple-red
Background – blue-violet
Nuclei – brown

Notes

Methyl violet may be used instead of crystal violet if preferred.
Although levulose syrup (fructose syrup or high fructose corn syrup) is specified it is likely that Highman’s gum syrup would be preferable as it inhibits leaching of the dye.
Although bismarck brown is metachromatic (yellow metachromasia), it is used here as a basic dye for staining nuclei.

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, p.451.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.

March 30, 2023

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Jurgens’ Crystal Violet for Amyloid

By Carmen WongAmyloid, Metachromasia, Protocols, Stain Target, Stain Type

Protocol

Jurgens' Crystal Violet

for Amyloid

steps

materials

print

Materials

Crystal violet, 1% aqueous
Acetic acid, 0.5% – 1% aqueous
Highman’s gum syrup

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Cryostat sections usually show brighter metachromasia. Unmounted frozen sections may also be floated in each solution and mounted on a slide just before coverslipping.

Protocol

Bring sections to water via xylene and ethanol, except for cryostat and frozen sections.
Place into crystal violet solution for 2-5 minutes.
Rinse well with water and examine microscopically.
If necessary, differentiate in dilute acetic acid until amyloid is red and contrasts well with the tissue.
Wash very well in tap water, about 5 minutes.
Drain all water from the slide until just damp and mount with Highman’s medium.
Ring the coverslip to inhibit evaporation of the mounting medium and precipitation of the ingredients.

Expected Results

Amyloid – purple-red
Background – blue-violet
Nuclei – blue-violet

Notes

Methyl violet may be used instead of crystal violet if preferred.
Highman’s gum syrup is a modification of Apathy’s gum syrup and contains potassium acetate or sodium chloride to stop bleeding of the dye into the mounting medium.

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. 222.
Oxford University Press, London, England.

March 30, 2023

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Gram Churukian–Schenk for Gram Positive & Negative Bacteria

By Carmen WongBacteria, Gram Staining, Protocols, Stain Target, Stain Type

Protocol

Gram Churukian–Schenk

for Gram Positive & Negative Bacteria

steps

materials

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Materials

Stock basic fuchsin
Material Amount
Basic fuchsin 0.5 g
Distilled water 100 mL
Solution A
Crystal violet 10% in 2mL ethanol
Material Amount
Ammonium oxalate 1% aqueous 98 mL
Solution B
Material Amount
Iodine 2 g
Potassium iodide 4 g
Distilled water 400 mL
Solution C
Material Amount
Absolute ethanol 1 volume
Acetone 1 volume
Solution D
Material Amount
Stock basic fuchsin 5 mL
Distilled water 45 mL
Solution E
Material Amount
Picric acid 0.1 g
Acetone 100 mL
Solution F
Material Amount
Acetone 1 Volume
Xylene 1 volume

Material	Amount
Basic fuchsin	0.5	g
Distilled water	100	mL

Material	Amount
Ammonium oxalate 1% aqueous	98	mL

Material	Amount
Absolute ethanol	1	volume
Acetone	1	volume

Material	Amount
Stock basic fuchsin	5	mL
Distilled water	45	mL

Material	Amount
Picric acid	0.1	g
Acetone	100	mL

Material	Amount
Acetone	1	Volume
Xylene	1	volume

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 in solution A for 2 minutes.
Rinse with tap water.
Place in solution B for 1 minute.
Rinse well with tap water.
Blot the slide, but not the tissue.
Decolorise with solution C until no more blue floods off.
Wet section with solution D then apply for 1 minute.
Rinse with distilled water.
Blot the slide, but not the tissue.
Place in acetone for 3 seconds.
Differentiate in solution E for 10 seconds.
Quickly dip a few times in solution F.
Clear with xylene and mount with a resinous medium.

Expected Results

Gram positive bacteria – blue
Nocardia and actinomyces – blue, or blue and red
Gram negative bacteria – red
Nuclei, Elastic, Paneth cells – red
Background – yellow

Notes

Picric acid should be handled with care. Solution E may be made by taking 12 mL of a saturated solution of picric acid in ethanol and diluting to 1 liter with acetone.
Basic fuchsin homologues with CI numbers of 42500 (pararosanilin) or 42510 (rosanilin) were specified. It was also noted that new fuchsin (CI 42520) was satisfactory, but not recommended because it was not certified by the Biological Stain Commission.
The authors note that sections must not be allowed to dry out after being stained with basic fuchsin. Doing so makes it difficult or impossible to properly differentiate the red counterstain.

Safety Note

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

References

Churukian, C. J. & Schenk, E. A. (1982)
A method for demonstrating Gram-positive and Gram-negative bacteria.
Journal of Histotechnology, v.5, No.3, p.127

March 30, 2023

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Gram Weigert for Fibrin and Gram Positive Bacteria

By Carmen WongBacteria, Fibrin, Gram Staining, Protocols, Stain Target, Stain Type

Protocol

Gram Weigert

for Fibrin and Gram Positive Bacteria

steps

materials

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Materials

Eosin

Material	Amount
Eosin Y ws	5	g
Distilled water	100	mL

Crystal violet

Material	Amount
Crystal violet	1	g
Distilled water	100	mL

Gram’s iodine

Material	Amount
Iodine	2	g
Potassium iodide	4	g
Distilled water	400	mL

Aniline-Xylene

Material	Amount
Aniline	1	volume
Xylene	1	volume

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 in eosin solution for 5 minutes.
Rinse with tap water.
Place in crystal violet 1 minute.
Rinse with tap water.
Flood with Gram’s iodine for 1 minute.
Rinse with tap water.
Gently blot the section, being careful not to damage it.
Decolorise the section with aniline-xylene.
Rinse with several changes of xylene to remove all aniline.
Mount with a resinous medium.

Expected Results

Gram positive bacteria – blue
Fibrin – blue
Background – pink

Notes

Control the differentiation with aniline-xylene microscopically. To examine, place the section in xylene to stop dye removal. Return to aniline-xylene if more differentiation is needed. Stop differentiation when the target element has good contrast.
Increasing the aniline content of the aniline-xylene will increase the speed of dye removal. Decreasing it will slow dye removal.
If the background is not pink enough, increase the time in eosin, stain in eosin at elevated temperature, or increase the eosin concentration.
The eosin counterstain may be omitted entirely if wished.

Safety Note

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

References

Culling, C.F.A., (1963)
Handbook of histopathological techniques, 2nd ed.
Butterworths, London.
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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Jones’ Impregnation for Basement Membranes

By Carmen WongAldehydes, Carbohydrates, Metal Impregnation, Metal Impregnation, Silver, Protocols, Stain Target, Stain Type

Protocol

Jones' Impregnation

for Basement Membranes

steps

materials

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Materials

Periodic acid, 0.5% aqu.
Neutral red, 1% aqu.
Yellow gold chloride, 0.2% aqu.
Light green SFy, 0.2% in 0.2% acetic acid, or progressive hemalum and eosin
Sodium thiosulfate, 2.5% aqu.
Stock Methenamine silver
Material Amount
Methenamine, 3% aqu. 100 mL
Silver nitrate, 5% aqu. 5 mL
Shake until the precipitate redissolves. Silvering of the container indicates deterioration.
Working Methenamine silver
Material Amount
Stock Methenamine silver 50 mL
Borax, 5% aqu. 5 mL
Make just before use and preheat to 50°C.

Material	Amount
Methenamine, 3% aqu.	100	mL
Silver nitrate, 5% aqu.	5	mL

Material	Amount
Stock Methenamine silver	50	mL
Borax, 5% aqu.	5	mL

Tissue Sample

3µ paraffin sections of neutral buffered formalin or Bouin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended. Thinner sections are to be preferred. This method gives excellent results with deplasticized methyl methacrylate sections at 1µ.

Protocol

Bring sections to water via xylene and ethanol.
Oxidize with 0.5% periodic acid for 15 minutes.
Rinse well with tap water.
Rinse with distilled water.
Treat with methenamine silver solution at 50&degC. until impregnated (up to 3 hours)
Wash with distilled water.
Tone with 0.2% gold chloride solution for 2 minutes.
Rinse with distilled water.
Fix in 2.5% sodium thiosulfate for 3 minutes.
Wash well with running tap water.
Counterstain with light green, neutral red or a light H&E.
Rinse with tap water.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Basement membranes – black
Oxidisable carbohydrates – black
Background – as counterstained

Notes

In order to see the basement membranes on edge it is necessary to use the thinnest sections possible, especially for glomeruli.
Methenamine is also known as hexamethylenetetramine and hexamine.
Borax is sodium tetraborate.
Toning is a variable step. Untoned sections give dark brown material on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black material on a pale grey-brown background. Others tone longer (a few minutes) to produce black material on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.
This method depends on a similar principle to the periodic acid Schiff reaction, but in which the aldehydes produced by oxidation reduce a silver solution instead of combining with Schiff’s reagent to form a red compound. Consequently, those materials which are red in a PAS will be black in Jones’ stain, i.e. it is not specific for basement membranes but will demonstrate any carbohydrates which can be oxidised to aldehydes.
Hayashi, Tome and Shimosato recommended that, after oxidation with periodic acid, thiosemicarbazide should be applied to the section. Thiosemicarbazide has the formula H₂NNHCSNH₂. The hydrazine group (H₂NNH-) combines with any aldehydes generated by periodic acid oxidation. The thiocarbamyl group (-CSNH₂) is a more powerful reducing agent than the aldehydes it replaces and reduces the methenamine silver solution more rapidly and with higher contrast.Immediately following step 3:
- Place sections in 1% aqueous thiosemicarbazide for 10 minutes.
- Wash well with tap water, and carry on from step 4.
It is well known that metallic azides can be explosive. However, thiosemicarbazide is not a simple metallic azide. The MSDS says:
- Flash Point: n/a
- Lower Explosive Limit: n/a
- Upper Explosive Limit: n/a
- Unusal Fire and Expl.rds: none identified

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. 5.
Oxford University Press, London, England.
Hayashi, I., Tome, Y. and Shimosato, Y., (1989)
Thiosemicarbazide used after periodic acid makes methenamine silver staining of renal glomerular basement membranes faster and cleaner.
Stain Technology, v 64, p 185.

March 30, 2023

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DMAB-Nitrite for Tryptophan

By Carmen WongAmyloid, Protocols, Stain Target

Protocol

DMAB-Nitrite for Tryptophan

steps

materials

print

The DMAB-nitrite histochemical method is a simple and very highly selective, almost specific, method for the amino acid, tryptophan. A blue product obtained with this technique is invariably accepted as proof that the blue stained material contains tryptophan.

DMAB is p-dimethylaminobenzaldehyde, also known as 4-dimethylaminobenzaldehyde, 4-Formyl-N,N-dimethylaniline and N,N-Dimethyl-4-formylaniline.

Tryptophan

DMAB

p-Dimethylaminobenzaldehyde

Beta Carboline

Product: Beta Carboline

The leftmost structural formula above is of tryptophan, the central one is of DMAB, and the rightmost formula is of the initial reaction product that is produced by them. This reaction product is then oxidized with potassium nitrite to form an easily seen, blue colored compound of unspecified structure. It should be noted that formaldehyde and glyceraldehyde can also participate in similar reactions, so if this method is contemplated then formalin fixation should be kept short. The common overnight fixation in 10% formalin variants still permits the technique to give a positive result.

Materials

p-Dimethylaminobenzaldehyde, 5% in concentrated hydrochloric acid
Sodium nitrite (NaNO2), 1% in concentrated hydrochloric acid

Tissue Sample

15µ paraffin sections of tissues fixed up to 24 hours in one of:

1% trichloracetic acid in 80% ethanol
10% sulphosalicylic acid
10% neutral buffered formalin (6 hours preferred)

Protocol

Bring sections to ethanol via xylene and ethanol and allow to just become dry.
Place into DMAB solution for 1 minute
Place in sodium nitrite solution for 1 minute.
Rinse with tap water for 30 seconds.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Tryptophan – blue

Notes

Positive results should be seen in fibrin and fibrinoid, amyloid, Paneth cell granules, peptic cell granules, zymogen granules, muscle, neurokeratin and hair root sheath. Of these, only fibrin, including fibrinoid, and amyloid are extracellular materials which could be confused.
When used to confirm that a material is amyloid, the positive material may be differentiated from fibrin by dye staining methods, i.e. a positive DMAB-nitrite stain with a congo red stain also positive would identify the stained material as amyloid with a high degree of certainty, as fibrin is not stained with that dye.

Safety Note

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

References

Adams, C.W.M., (1957),
A p-dimethylaminobenzaldehyde-nitrite method for the histochemical demonstration of tryptophane and related compounds.,
Journal of Clinical Pathology, v 10, page 56-62.
Pearse, A.G.E. (1968).
Histochemistry: Theoretical and Applied, Ed. 3, Volume 1.
Churchill Livingstone, London, England.
Davenport, H.A.. (1960).
Histological and Histochemical Technics,
W. B. Saunders, Philadelphia, USA.

March 30, 2023

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