Foot's Impregnation
for Reticulin

Solutions

Reagent Var I Var II Var III Var 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)
Iodine
Potassium iodide
Distilled Water
6 g
4 g
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 Foot's tannin solution
Yellow gold chloride
Mercuric chloride
Distilled Water
0.2 g
0.5 g
100 mL
Tannic acid
Ammonium bromide
Strong formalin
Distilled Water
0.15 g
3.5 g
5 mL
100 mL
Foot's ammoniacal silver – Var 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.
 
Foot's ammoniacal silver – Var 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.
 
Foot's ammoniacal silver – Var 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.
 
Foot's ammoniacal silver – Var 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
5µ 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.

Method

# Step Var I Var II Var III Var IV

1

Bring sections to water via xylene and ethanol.
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
a. Place in Foot's tannin solution at 37°C
b. Place in 0.1% ammonium hydroxide
48 hrs


15 min
30 sec




9 Rinse with distilled water 5 sec 5 sec
10 Foot's ammoniacal silver solution, VAR I
Foot's ammoniacal silver solution, VAR II, 2 changes, each for
Foot's ammoniacal silver solution, VAR III at 45°C
Foot's ammoniacal silver solution, VAR IV at 45°C
30 min



5 min



15 min



15 min
11 Rinse with distilled water 5 sec 5 sec 5 sec
12 Reduce with 10% formalin
Reduce with 20% formalin
Flood with 1% formalin twice
30 min
——

3 min—

2 min—


10 sec
13 Rinse with distilled water
Wash off with tap water


5 sec

5 sec
14 Tone with 1% yellow gold chloride
Tone with Foot's gold sublimate
Tone with 0.2% yellow gold chloride
1 hr


3 min


2 min


2 min
15 Wash with distilled water
Wash with tap water
1 min
1 min


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
Place in Harris' alum hematoxylin
Place in neutral red
1 min




3 min


1 min
19 Wash with tap water 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

 

Expected results

Notes

  1. 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.
  2. 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.
  3. 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 three methods were taken from Gray.
  4. 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).
  5. 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..

 

Reference
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.

 


 

 

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