Methods & Techniques
For Histologists and Immunohistochemists		  

von Kossa Staining Protocol for Calcium

 

 

 

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 ---------------- 5 g

      Distilled water  -------------------- 100 ml

 

0.1% Nuclear Fast Red Solution:

      Nuclear fast red ------------------- 0.1 g

      Aluminum sulfate------------------ 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: 

  1. Deparaffinize paraffin sections and hydrate to water.

  2. Rinse in several changes of distilled water.

  3. 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).

  4. Rinse in several changes of distilled water.

  5. Remove un-reacted silver with 5% sodium thiosulfate for 5 minutes.

  6. Rinse in distilled water.

  7. Counterstain with nuclear fast red for 5 minutes.

  8. Rinse in distilled water.

  9. Dehydrate through graded alcohol and clear in xylene.

  10. Coverslip using permanent mounting medium.

Results:

Calcium salts ------------------------ black or brown-black

Nuclei -------------------------------- red

Cytoplasm --------------------------- pink

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Positive Controls:

 

      16-18 days mouse embryo, calcium containing tissues or undecalcified bone.

 

Notes:

 

  1. UV light usually gives stronger reaction so the calcium salts are often stained black. The regular 60-100 watt light bulb usually gives weaker reaction so the calcium salts are often stained brown-black.

  2. Oxalate salts are usually believed to give a negative von Kossa staining.

  3. A negative control may be needed when there is any doubt that the resulting black deposits are calcium. This is done by treating a test slide in 10% formic acid for 10 minutes prior to step 3. The test slide should show negative reaction.

 

 

References:

 

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