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.

Foot’s Impregnation for Reticulin