Foot's Impregnation
for Reticulin
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.