Although the Von Kossa technique is said to demonstrate calcium, it actually demonstrated the anion rather than calcium itself. This is usually carbonate or phosphate with a cation of calcium, so the method is commonly considered to demonstrate that material. However, urates are also blackened, and it should always be born in mind that some other anion could be being demonstrated.
Solutions Neutral red
Silver nitrate, 5% in distilled water
Sodium thiosulphate, 5% in distilled water
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable.
Other fixatives are likely to be satisfactory, although those containing strong acids may remove some calcium deposits..
Bring sections to distilled water via xylene and ethanol.
Place the sections flat on a suitable support (glass rods in a Petri dish).
Flood with silver solution.
Expose to strong, direct sunlight for 15-60 minutes. If this is not possible, an ultra-violet light held at a distance
of about 5 cm is usually suitable.
Wash well with distilled water to remove all traces of silver nitrate.
Place into thiosulphate for 5 minutes.
Wash well with running tap water for 5 minutes.
Counterstain with neutral red.
Dehydrate with ethanols.
Clear with xylene and mount with a resinous medium.
Calcium deposits – dark brown or black
Nuclei – red
Time of exposure to light may be determined visually. The silver solution may be removed when
deposits are seen to be distinctly black.
To increase the confidence that the blackened material is calcium, a duplicate section may be
treated with 0.5% aqueous hydrochloric acid for a few minutes. Calcium is removed.
Pearse recommended 0.1M citrate buffer pH 4.5 applied for 20 minutes for this purpose.
Culling C.F.A., (1974) Handbook of histopathological and histochemical techniques Ed. 3
Butterworth, London, UK.