Picro-Mallory

for Fibrin – Shorter Version

steps

materials

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This version of the picro-Mallory is relatively uncomplicated and is suitable for a routine pathology laboratory. Proper fixation is still essential and minimalist formalin fixation with quick processing should be avoided as it will give disappointing results with poorly stained erythrocytes. Originally, extended mercury fixation, thorough processing, degreasing and refixing in picro-mercuric-ethanol were specified. Although fixation in formol sublimate (or B5) is preferred for this method as well, adequate results can be obtained with overnight formalin fixation and overnight processing. Refixation of sections in Bouin’s fluid at 56°C for an hour or so overcomes some of the deficiencies in fixation, but results are still inferior to those obtained by the full procedure.

Materials

An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
Picro-orange
Material Amount
Picric acid, sat. in 80% ethanol 100 mL
Orange G 0.2 g
Yellow differentiator
Material Amount
Picro-orange 30 mL
Ethanol, 80% 70 mL
Red stain
Material Amount
Acid fuchsin 1 g
Acetic acid, glacial 1 mL
Distilled water 99 mL
Red differentiator
Material Amount
Phosphotungstic acid 1 g
Distilled water 100 mL
Blue stain
Material Amount
Methyl blue 2 g
Acetic acid, glacial 2 mL
Distilled water 98 mL

Material	Amount
Picric acid, sat. in 80% ethanol	100	mL
Orange G	0.2	g

Material	Amount
Picro-orange	30	mL
Ethanol, 80%	70	mL

Material	Amount
Acid fuchsin	1	g
Acetic acid, glacial	1	mL
Distilled water	99	mL

Material	Amount
Phosphotungstic acid	1	g
Distilled water	100	mL

Material	Amount
Methyl blue	2	g
Acetic acid, glacial	2	mL
Distilled water	98	mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate (or B5) overnight. Paraffin process overnight. Avoid rapid fixation with formalin and short processing, as this produces tissues that stain poorly even with the secondary fixation specified. Sections should be 3-5 µ thick.

Protocol

Bring sections to water via xylene and ethanol.
Remove mercury pigment with the iodine-thiosulphate sequence.
Place into Bouin’s fluid at 56°C for 1 hour.
Rinse well in distilled water.
Stain nuclei with an acid resistant nuclear stain.
Rinse with 95% ethanol.
Place in picro-orange for 2 minutes.
Without rinsing, place in the red stain for 5 minutes.
Rinse with distilled water.
Dip into yellow differentiator.
Rinse in distilled water.
Differentiate with the red differentiator for 5 minutes.
Rinse with distilled water.
Place in the blue stain for 2 minutes.
Rinse briefly with 1% aqueous acetic acid.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Fibrin – clear red
Fibrinoid – varies from red to orange
Erythrocytes – orange
Connective tissue – blue

Notes

If possible, the extended fixation and slow processing specified for the longer version should be used. Degreasing and refixation will also improve results if time allows.

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.

March 30, 2023

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Yellowsolve I for Fibrin

By Carmen WongFibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Yellowsolve Staining

Protocol

Yellowsolve I

for Fibrin

steps

materials

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Materials

Celestine blue-hemalum nuclear stain, or similar.
Trichlorethylene
Tetrachlorethylene
Solution A
Material Amount
Phloxine B 0.5 g
Calcium chloride 0.5 g
Distilled water 100 mL
Solution B
Material Amount
Lissamine flavine FF to saturation
2-Ethoxyethanol 50 mL
Ethyl phosphate 50 mL
Optional – an elastic fibre stain: Orcein or Weigert’s iron resorcin fuchsin, or variant.

Material	Amount
Phloxine B	0.5	g
Calcium chloride	0.5	g
Distilled water	100	mL

Material	Amount
Lissamine flavine FF	to saturation
2-Ethoxyethanol	50	mL
Ethyl phosphate	50	mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are likely suitable. It should be noted, however, that the authors of this method favoured extended fixation in formal sublimate of up to 10 days for fibrin. This fixative is now deprecated due to its mercuric chloride content. With other staining methods, pretreatment of sections with Bouin’s fluid for an hour at 60°C can compensate for the lack of mercury fixation to a large degree.

Protocol

Dewax sections with xylene.
Place in trichlorethylene for 24 to 48 hours.
Bring sections to water.
Stain elastic fibres if desired.
Stain nuclei with the celestine blue-hemalum sequence.
Wash well with water.
Place in solution A for 30 minutes.
Rinse with 2-ethoxyethanol then with tetrachlorethylene.
Place into tetrachlorethylene overnight.
Rinse with 2-ethoxyethanol.
Differentiate with solution B, controlling microscopically.
Rinse with 2-ethoxyethanol.
Clear in xylene and mount with a synthetic resinous medium.

Expected Results

Fibrin – red
Background – yellow
Nuclei – black
Acidophil cytoplasmic inclusions – red

Notes

2-Ethoxyethanol is also known as cellosolve or ethylene glycol monoethyl ether, and has the formula CH₃CH₂OCH₂CH₂OH.
Tetrachlorethylene has the formula Cl₂C=CCl₂ or C₂Cl₄
Trichlorethylene has the formula ClHC=CCl₂ or C₂HCl₃
Both trichlorethylene and tetrachlorethylene should be used in a fume hood.

Safety Note

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

References

Lendrum A C, Fraser D S, Slidders W and Henderson R. (1962)
Studies on the character and staining of fibrin.
Journal of clinical pathology, v. 15, p. 401.

March 30, 2023

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Shum & Hon’s PTAH

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

Protocol

Shum & Hon's PTAH

steps

materials

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Materials

Material	Amount	Function
Hematein,	0.8 g.	Dye
Phosphotungstic acid	9 g	Mordant
Distilled water	1 L	Solvent

Compounding procedures

Combine the hematein, phosphotungstic acid and 10 mL of the water and grind them to a paste with a pestle and mortar.
Wash the contents into a beaker with the rest of the water.
Bring the solution to a boil, then cool and filter.

Protocol

Bring sections to water with xylene and ethanol.
Perform a Mallory bleach using 0.25% potassium permanganate.
Rinse well with water.
Place into the PTAH solution for 12 – 24 hours at room temperature.
Rinse well with water.
Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Nuclei, erythrocytes, fresh fibrin, muscle striations – blue
Background – red

Notes

Observe the colour of the sludge while grinding as a good sample of hematein will be chocolate brown. If the sludge is pale, the final solution may not be satisfactory.
The staining may be done in an oven at about 60°C for a few hours, but the results are often less satisfactory.
Many substances have been stated to be stained blue. Sometimes the red staining overshadows blue staining, and the section may need to be washed to remove excess red.

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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Foot’s Impregnation for Reticulin

By Carmen WongMetal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type

Protocol

Foot's Impregnation

for Reticulin

steps

materials

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Materials

Material	Variation I	Variation II	Variation III	Variation IV
Lugol’s iodine	+	+	+	+
Sodium thiosulphate, 1% aqu.	+	+	+	+
Sodium thiosulphate, 5% aqu.	+	+	+	+
Potassium permanganate, 0.25% aqu.	+	+	+	+
Oxalic acid, 5% aqu.	+	+	+	+
Strong ammonium hydroxide (s.g. 0.88)	+	+	+	+
Ammonium hydroxide, 0.1% aqu	–	+	–	–
Foot’s tannin sol.	–	+	–	–
Silver nitrate, aqu.	2% & 10%	1% & 10%	10%	10%
Formalin, aqu.	5%	20%	20%	1%
Yellow gold chloride, aqu.	1%	–	0.2%	0.2%
Foot’s gold sublimate	–	+	–	–
Alkali, aqu.	NaOH 40%	KOH 40%	LiC0₃ sat.	Na₂CO₃
Weigert’s 1903 iron hematoxylin	+	–	–	–
Harris alum hematoxylin	–	–	+	–
van Gieson’s picro-fuchsin	+	–	+	–
Neutral red, 1% aqu.	–	–	–	+

Lugol’s Iodine (see note)

Material	Amount
Iodine	6	g
Potassium iodide	4	g
Distilled Water	100	mL

Mix the iodine and potassium iodide in a 200 mL flask. Add 10 mL of the water. When the iodine has dissolved make up to 100 mL with distilled water.

Foot’s gold sublimate

Material	Amount
Yellow gold chloride	0.2	g
Mercuric chloride	0.5	g
Distilled Water	100	mL

Foot’s tannin solution

Material	Amount
Tannic acid	0.15	g
Ammonium bromide	3.5	g
Strong formalin	5	mL
Distilled Water	100	mL

Preparation of Foot’s Ammoniacal Silver

Variation I

Place 25 mL of 10% silver nitrate in a flask.
Add 1 mL of 40% sodium hydroxide.
Add ammonium hydroxide by drops until the precipitate is almost dissolved.
Dilute to 100 mL with distilled water.

Variation II

Place 10 mL of 1% silver nitrate in a flask.
Add 0.1 mL of 40% potassium hydroxide.
Add ammonium hydroxide by drops until the precipitate is almost dissolved.
Dilute to 100 mL with distilled water.

Variation III

Place 10 mL of 10% silver nitrate in a flask.
Add 10 mL of saturated aqueous lithium carbonate.
Let the precipitate settle, then decant the supernatent.
Wash, allow to settle, and decant several times.
Add 25 mL distilled water to the precipitate.
Add ammonium hydroxide by drops until the precipitate is almost dissolved.
Dilute to 100 mL with distilled water and filter.

Variation IV

Place 10 mL of 10% silver nitrate in a flask.
Add 40 mL of aqueous sodium carbonate (see note for concentration).
Let the precipitate settle, then decant the supernatent.
Wash, allow to settle and decant several times.
Add ammonium hydroxide by drops until the precipitate is almost dissolved.

Tissue Sample

5 µ paraffin sections of Zenker fixed tissue are suitable. Other fixatives may be satisfactory. Carleton & Leach say that Var IV is suitable for 10% formalin, Zenker, Bouin or Helly fixed tissue. In all cases a section adhesive is recommended.

Protocol

Step		Variation I	Variation II	Variation III	Variation IV
1	Bring sections to water via xylene and ethanol (all variations).
2	Place in Lugol’s iodine.	5 min	5 min	5 min	5 min
3	Bleach in 1% sodium thiosulphate.	30 sec	30 sec	30 sec	30 sec
4	Oxidise in 0.25% potassium permanganate.	5 min	5 min	5 min	5 min
5	Bleach with 5% oxalic acid.	10 min	10 min	10 min	10 min
6	Wash well with tap water.	5 min	5 min	5 min	5 min
7	Rinse with distilled water.	5 sec	5 sec	5 sec	5 sec
8	Place in 2% silver nitrate.	48 hrs	—	—	—
	Place in Foot’s tannin solution at 37°C.	—	15 min	—	—
	Place in 0.1% ammonium hydroxide.	—	30 sec	—	—
9	Rinse with distilled water.	5 sec	5 sec	—	—
10	Place in Foot’s ammoniacal silver solution (Variation I).	30 min	—	—	—
	Place in Foot’s ammoniacal silver solution (Variation II), 2 changes, each for:	—	5 min	—	—
	Place in Foot’s ammoniacal silver solution (Variation III) at 45°C.	—	—	15 min	—
	Place in Foot’s ammoniacal silver solution (Variation IV) at 45°C.	—	—	—	15 min
11	Rinse with distilled water.	5 sec	5 sec	5 sec	—
12	Reduce with 10% formalin.	30 min	—	—	—
	Reduce with 20% formalin.	—	3 min	2 min	—
	Flood with 1% formalin twice.	—	—	—	10 sec
13	Rinse with distilled water.	—	—	5 sec	—
	Wash off with tap water.	—	—	—	5 sec
14	Tone with 1% yellow gold chloride.	1 hr	—	—	—
	Tone with Foot’s gold sublimate.	3 min	—	—	—
	Tone with 0.2% yellow gold chloride.	—	—	2 min	2 min
15	Wash with distilled water.	1 min	1 min	—	—
	Wash with tap water.	—	—	—	2 min
16	Place in 5% sodium thiosulphate.	—	3 min	2 min	2 min
17	Wash with tap water.	5 min	5 min	5 min	5 min
18	Place in Weigert’s iron hematoxylin.	1 min	—	—	—
	Place in Harris’ alum hematoxylin.	—	—	3 min	—
	Place in neutral red.	—	—	—	1 min
19	Wash with tap wate.	5 min	5 min	5 min	1 min
20	Place in van Gieson’s picro-fuchsin.	30 sec	—	45 sec	—
22	Dehydrate with ethanol, clear with xylene and mount in a resinous medium (all variations).

Expected Results

Reticulin fibres – black
Nuclei – as counterstained
Background – grey

Notes

Ensure that the ammonium hydroxide is fresh and full strength. Keep well stoppered when not in use. After removing the amount required, immediately restopper the bottle.
Improperly made ammoniacal silver solutions can affect the quality of the impregnation. There should be a faint, persistent opalescence, with only a faint smell of ammonia.
The formula for Lugol’s iodine may be an error. The amounts may have been reversed,
i.e. It may be 4 g iodine and 6 g potassium iodide. Please refer to the page for
Lugol’s iodine and compare Gray and Lee. The details for these methods are from Gray.
The instructions for ammoniacal silver solution Var IV says to add 40 mL of sodium carbonate to the silver nitrate solution. The sodium carbonate should be made as a 5% solution of the anhydrous salt or as an 11% solution of the dodecahydrate (12H₂O).
1%, 10% and 20% formalin solutions are made by diluting strong formalin appropriately with water, i.e.:
- 1% is 1 mL strong formalin plus 99 mL water,
- 10% is 10 mL strong formalin plus 90 mL water, and
- 20% is 20 mL strong formalin plus 80 mL water.

Safety Note

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

References

Gray, Peter. (1954), (for Var I, II & III)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Carleton, H M, and Leach, E H, (1938), (for Var IV)
Histological technique., Ed. 2.
Oxford University Press, London, England.

March 30, 2023

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Friedlander’s Alum Hematoxylin

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

Protocol

Friedlander's Alum Hematoxylin

steps

materials

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Materials

Material	Amount	Function
Hematoxylin	6 g	Dye
Potassium alum	6 g	Mordant
Distilled water	300 mL	Solvent
95% ethanol	300 mL	Solvent
Glycerol	300 mL	Stabiliser

Compounding procedures

Dissolve the Alum in the water.
Dissolve the hematoxylin in the ethanol.
Combine, and add glycerol.

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for an appropriate time.
Rinse well with water.
Differentiate with acid ethanol if necessary.
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

This is probably a regressive formula.
The staining time should be determined by trial.
Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
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

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.

March 30, 2023

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Gridley’s Stain for Fungi

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

Protocol

Gridley's Stain

for Fungi

steps

materials

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Materials

Schiff’s reagent
Aldehyde fuchsin
Chromic acid
Material Amount
Chromium trioxide 5 g
Distilled water 500 mL
Bleach
Material Amount
Sodium metabisulfite 5 g
Distilled water 500 mL
Metanil yellow
Material Amount
Metanil yellow 1 g
Distilled water 400 mL
Acetic acid, glacial 2 drops

Material	Amount
Chromium trioxide	5	g
Distilled water	500	mL

Material	Amount
Sodium metabisulfite	5	g
Distilled water	500	mL

Material	Amount
Metanil yellow	1	g
Distilled water	400	mL
Acetic acid, glacial	2	drops

Tissue Sample

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

Protocol

Bring sections to water via xylene and ethanol.
Place in chromic acid for 1 hour.
Wash well with tap water.
Treat with the metabisulfite bleach for 1 minute.
Wash well with tap water.
Rinse with distilled water.
Place in Schiff’s reagent for 20 minutes.
Wash well with tap water.
Rinse with 70% ethanol.
Place in aldehyde fuchsin 30 minutes.
Rinse off excess with 95% ethanol.
Wash well with tap water.
Counterstain with metanil yellow for 1 minute.
Rinse well with distilled water.
Dehydrate, clear and mount in a resinous medium.

Expected Results

Fungi – purple
Background – yellow

Notes

At step 2, a 10% solution of chromic acid applied for 10 minutes will give similar results.

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. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, 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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Gadsdon’s Alum Hematoxylin

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

Protocol

Gadsdon's Alum Hematoxylin

steps

materials

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This formula is by Prof. Derek Gadsdon at the Sheffield Children’s Hospital, UK.

Materials

Material	Amount	Function
Hematoxylin	5.5 g	Dye
Potassium alum	60 g	Mordant
Distilled water	610 mL	Solvent
100% ethanol	100 mL	Solvent
Glycerol	300 mL	Stabiliser
Sodium iodate	0.5 g	Oxidant
Glacial acetic acid	20 mL	Acidifier

Compounding procedures

Dissolve the hematoxylin in 100 mL ethanol.
Dissolve the Alum in 600 mL water.
Dissolve the sodium iodate in 10 mL water.
Combine the hematoxylin and Alum solutions. Mix well.
Add the sodium iodate solution. Mix well.
Add the acetic acid. Mix well.
Leave overnight.
Add the glycerol. Mix well.
Filter before use.
The solution may be used immediately.
It is stable at room temperature for at least 6 months.

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for the appropriate time.
Rinse well with water.
Differentiate with acid ethanol if using regressively.
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

This solution may be used regressively or progressively.
- Regressively, staining time is 6 minutes.
- Progressively, staining time is 3 minutes.
- Frozen sections will stain in 1 minute.
As with many strong formulations mucins may stain blue.
Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
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

Histopathology laboratory, Red Cross Children’s Hospital.
Gadsdon’s Haematoxylin Method
Histonet communication, Nov, 1996
Jim Elsam, HTEQA Services.
Gadsdon’s Hx
Histonet communication, Nov, 1996

March 30, 2023

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Gage’s Alum Hematoxylin

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

Protocol

Gage's Alum Hematoxylin

steps

materials

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Materials

Material	Amount	Function
Hematoxylin	1 g	Dye
Potassium alum	40 g	Mordant
Distilled water	1 L	Solvent
95% ethanol	20 mL	Solvent
Chloral hydrate	20 g	Oxidant

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for an appropriate time.
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

The formula indicates the solution is progressive.
As no oxidising agent is present the solution needs ageing.
The appropriate time should be determined by trial, but 5-10 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

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.

March 30, 2023

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Galigher’s Alum Hematoxylin

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

Protocol

Galigher's Alum Hematoxylin

steps

materials

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This solution was recommended as a substitute for Harris’ Alum hematoxylin.

Materials

Material	Amount	Function
Hematoxylin	5 g	Dye
Ammonium alum	3 g	Mordant
Distilled water	500 mL	Solvent
100% ethanol	500 mL	Solvent
Mercuric oxide	6 g	Oxidant

Compounding procedures

Combine the water and ethanol.
Add the Alum and dye, and bring to a boil.
Add the mercuric oxide, then simmer for 20 minutes.
Restore the volume to 1 L with 50% ethanol.
Cool, and filter through double thickness filter paper.
Store in a tightly stopper bottle.
The solution may be used immediately, and is stable for about six months.

Protocol

Bring sections to water with xylene and ethanol.
Place into the staining solution for an appropriate time.
Rinse well with water.
Differentiate with acid ethanol if necessary.
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

Mercuric salts are toxic and no longer recommended for oxidation of hematoxylin. Ensure that discarded solution is disposed of as mercury contaminated, or substitute 0.5 grams sodium iodate as the oxidant.
Staining capability may continue to improve for a short while.
The appropriate staining time should be determined by trial.
Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
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

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

March 30, 2023

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Gallego’s Carbol Fuchsin for Nuclei

By Carmen WongDye Type, Hematoxylin Alternatives, Protocols, Stain Type

Protocol

Gallego's Carbol Fuchsin

for Nuclei

steps

materials

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Materials

Solution A

Material	Amount
Carbol fuchsin	2	mL
Distilled water	98	mL

Solution B

Material	Amount
Formalin, conc. (40%)	1	mL
Distilled water	99	mL

Tissue Sample

5 µ 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 solution A for 5 minutes.
Wash with water.
Place into solution B for 5 minutes.
Wash with water.
Stain with a contrast stain.
Dehydrate with ethanol.
Clear with xylene and mount with a resinous medium.

Expected Results

Nuclei – blue-black
Cytoplasm – as counterstain

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:
Gallego, (1919)
Trabajos del Laboratorio de investigaciones biológicas de la
Universidad de Madrid,
v. 17, pp.95
And:
Biot, (1910)
Compte rendu de l’Association des anatomistes, Congrès de Lyon, pp.234

March 30, 2023

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