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von Kossa Staining Protocol for Calcium
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von Kossa Staining Protocol for Calcium

Prepared by


IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011


Silver ions are displaced from solution by carbonate or phosphate ions, due to their respective positions in the electrochemical series.  The argentaffin reaction (see Masson Fontana), is photochemical in nature, and the activational energy is supplied from ultra violet light.  Since the demonstrable forms of tissue carbonate or phosphate ions are invariably associated with calcium ions, the method may be considered as demonstrating sites of tissue calcium deposition.  By selecting an appropriate counterstain, other tissue elements may be demonstrated as required, provided subsequent staining solutions used do not remove the precipitated silver ions.

Technical Points

1.  A known positive control section must be used to ensure correct demonstration has been achieved.


1.   Bring sections to distilled water.

2.   Flood slides with 5% aq silver nitrate

3.   Expose sections to bright sunlight, or UV lamp 20 mins

(Ultra violet light is dangerous to eyes and can cause blindness by damaging the retina).

4.   Wash well in distilled water

5.   Treat with 2% sodium thiosulphate 2 mins

6.   Wash in running tap water

7.   Rinse in distilled water

8.   Counterstain with 1% Neutral red 2 mins

9.   Rapidly dehydrate, clear and mount.



Reagent Formulae

1.   5% aq silver nitrate

2.   2% aq sodium thiosulphate

3.   Neutral red - acidified

       neutral red (CI 50040)        1.0 g
      distilled water                   100.0 ml
      gl acetic acid                    1.0 ml

Dissolve the dye in the distilled water.  Add the acid.  Mix well.  Filter into the reagent bottle.  Keeps well.


1.   von Kossa J,(1901),Beitr.path

2.   Bancroft, J.D. and Cook, H.C.  1984, Manual of Histological Techniques. Churchill Livingstone.

3.   Lillie, R.D.  1977, H.J.Conn's Biological Stains, 9th edition. Williams and Wilkins.

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