von Kossa Staining Protocol for Calcium
the protocol was taken from:
http://www.ihcworld.com/_protocols/special_stains/von_kossa.htm

I checked it and it works perfect

Description: This technique is for demonstrating deposits of calcium or calcium salt so it is not specific for the calcium ion itself. In this method, tissue sections are treated with a silver nitrate solution and the silver is deposited by replacing the calcium reduced by the strong light, and thereby visualized as metallic silver.

Fixation: formalin fixed, paraffin embedded tissue sections or alcohol fixed, frozen sections.

Solutions and Reagents:
1% Aqueous Silver Nitrate Solution:
Silver nitrate ------------------------- 1 g
Distilled water ---------------------- 100 ml
5% Sodium Thiosulfate:
Sodium thiosulfate ---------------- 1 g
Distilled water -------------------- 100 ml
0.1% Nuclear Fast Red Solution:
Nuclear fast red ------------------- 0.1 g
Ammonium sulfat------------------ 5 g
Distilled water ----------------------100 ml
Dissolve ammonium sulfate in water. Add nuclear fast red and slowly heat to boil and cool. Filter and add a grain of thymol as a preservative.

Procedure:
Deparaffinize paraffin sections and hydrate to water.
Rinse in several changes of distilled water.
Incubate sections with 1% silver nitrate solution in a clear glass coplin jar placed under ultraviolet light for 20 minutes (or in front of a 60-100 watt light bulb for 1 hour or longer).

Rinse in several changes of distilled water.
Remove un-reacted silver with 5% sodium thiosulfate for 5 minutes.

Rinse in distilled water.
Counterstain with nuclear fast red for 5 minutes.
Rinse in distilled water.
Dehydrate through graded alcohol and clear in xylene.

Coverslip using permanent mounting medium.

Results:
Calcium salts --------------------- black or brown-black
Nuclei -------------------------------- red
Cytoplasm ------------------------- pink

References:

Sheehan D, Hrapchak B, Theory and Practice of Histotechnology, 2nd Ed, 1980, pp 226-227, Battelle Press, Ohil.

Guy