Bensley & Bensley's
Impregnation for Reticulin
Solutions
Reagent | Var I | Var II |
Lugol's iodine | + | + |
Potassium permanganate, 1% aqu. | + | + |
Oxalic acid, 5% aqu. | + | + |
Sodium thiosulphate, 3% aqu. | + | + |
Tannic acid, saturated in ethanol, 95%. | + | – |
Strong ammonium hydroxide (s.g. 0.88) | + | + |
Ammoniated water | + | – |
Silver nitrate, aqu. | 1% | 2% |
Formalin, 20% aqu. | + | + |
Yellow gold chloride, 0.2% aqu. | + | + |
Sodium hydroxide, 40% aqu. | + | + |
Neutral red, 1% aqu. | + | + |
Lugol's iodine | |||
Iodine Potassium iodide Distilled Water |
1 g 2 g 300 mL |
Mix the iodine and potassium iodide in a 500 mL flask. Add 5 mL of the water. When the iodine has dissolved make up to 300 mL with distilled water. |
Ammoniacal silver – Var I |
Place 20 mL of 1% silver nitrate in a flask. Add 4 drops of 40% sodium hydroxide Add ammonium hydroxide by drops until the precipitate is almost dissolved. Dilute 1:10 with distilled water. |
Ammoniacal silver – Var II |
Place 20 mL of 2% silver nitrate in a flask. Add 3 drops of 40% sodium hydroxide Add ammonium hydroxide by drops until the precipitate is just dissolved. |
Tissue sample
Bensley & Bensley said that sections of tissue fixed in Zenker, Helly, ethanol or formalin are suitable.
Although they commented that Var I gave a complete impregnation of paraffin or celloidin embedded tissue, they recommended Var II for paraffin sections because of their tendency to detach from slides. A section adhesive is recommended.
Method
# | Step | Var I | Var II |
1 |
Bring sections to water via xylene and ethanol. |
||
2 | Oxidise in 1% potassium permanganate | 5 min | 5 min |
3 | Bleach with 5% oxalic acid | + | + |
4 | Place in Lugol's iodine | + | + |
5 | Bleach in 3% sodium thiosulphate | + | + |
6 | Wash with water | + | + |
7 | a. Place in tannic acid solution at 56°C b. Rinse with ammoniated distilled water Place in 2% silver nitrate |
5 min + — |
— — 16 hrs |
8 | Ammoniacal silver solution, VAR I, at 56°C Ammoniacal silver solution, VAR II, at RT |
10 min — |
— 30 min |
9 | Wash with water | + | + |
10 | Reduce with 20% formalin | + | 3 min |
11 | Wash with water | + | + |
12 | Tone with 0.2% yellow gold chloride | + | + |
13 | Wash with water | + | + |
14 | Place in 3% sodium thiosulphate | + | + |
15 | Wash with water | + | + |
16 | Place in neutral red | 1 min | 1 min |
17 | Wash with water | + | + |
18 | Dehydrate with ethanol, clear with xylene and mount in a resinous medium |
Expected results
Notes
Reference
Bensley, R R, and Bensley, S H, (1938
Handbook of Histological and Cytological technique.
The University of Chicago Press, Chicago, Illinois, USA.
Gray, Peter. (1954)
The Microtomist's Formulary and Guide.
Originally published by:– The Blakiston Co.
Republished by:– Robert E. Krieger Publishing Co.