This silver impregnation procedure labels the reticular fibers, which can be easily visualized in the lymphatic tissues and bone marrow.
Procedure
Samples are fixed and paraffin embedded. 8 µm thick sections are obtained and attached to a gelatin coated slide.
1.- 2x10 min in xylene
2.- 2x10 min in 100º ethanol
3.- 10 min in 96º ethanol
4.- 10 min in 80º ethanol
5.- 10 min in 50º ethanol
6.- 5 min in distilled H2O
7.- 1-2 min oxidation in 1 % potassium permanganate dissolved in distilled H2O
8.- Several rinses in distilled H2O
9.- 1 min clearing in 3 % potassium metabisulphite dissolved in distilled H2O
10.- 5 min rinse in abundant tap water
11.- 1 min in 2 % ferric alum dissolved in distilled H2O (fresh solution)
12.- 10 min rinse in abundant tap water
13.- 2 x 2 min rinses in distilled H2O
14.- 1 min in argentic solution (fresh solution).
The silver solution contains silver ammonium and distilled H2O (1:1). Silver ammonium: mix 10 ml of 10 % silver nitrate and 2 ml of 10 % potassium hydroxide. A precipitated is formed, which is dissolved with concentrate ammonium. After clearing the solution, more of 10 % of silver nitrate is added until precipitates are visible again. Then prepare the silver soluti
15.- 10 s in distilled H2O (one dip)
16.- Reduction in 10 % formaldehyde. The length of this step is determined experimentaly.
17.- 5 min in tap water
18.- 1 min en metabisulfito potásico al 1 %.
19.- 1 min in 1 % of sodium thiosufate.
20.- 5 min in tap water.
21.- 1 min in distilled H2O
22.- 5 min in 50º ethanol
23.- 5 min in 70º ethanol
24.- 5 min in 80º ethanol
25.- 5 min in 96º ethanol
26.- 2x5 min en 100ºn ethanol
27.- 2x5 min in xylene
28.- Mounting medium and coverslip.
Results
Reticular fibers: black.
Collagen: yellowish.
Notes
One critical point for a good labaling is the formaldehyde reduction step, which has to be determined experimentally.
The rinsing step before reduction has to be be very quick.
Products
Xylene
50º, 70º, 80º, 96º and 100º ethanol
Potassium permanganate
Potassium methabisulfite
Ferric alum
Silver nitrate
Potassium hydroxide
Concentrate ammonia
Sodium thiosulfate
Distilled H2O
Tap water
Mounting medium
Labware
Flasks, test tubes and containers
Staining dishes
Slides rackstaining dishes
Coverslips
