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.

Alcian blue stains acid mucosubstances and acetic mucins. Excessive amounts of non-sulfated acidic mucosubstances are seen in mesotheliomas, certain amounts occur normally in blood vessel walls but increase in early lesions of atherosclerosis. Strongly acidic mucosubstances will be stained blue, nuclei will be stained pink to red, and cytoplasm will be stained pale pink.

Combination of von Kossa and Alcian Blue staining will make both calcium and mucosubstances visible on the same tissue sections.

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

Alcian Blue Solution (pH 2.5):

      Alcian blue, 8GX -------------------- 1 g

      Acetic acid, 3% solution ----------- 100 ml

      Mix well and adjust pH to 2.5 using acetic acid.

0.1% Nuclear Fast Red Solution:

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

      Aluminum sulfate------------------ 5 g

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

      Dissolve aluminum 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). Note: If stain was weak or rinsed off in washing steps, it indicated the UV light was not strong enough. Longer staining is required for up to several hours. 
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. Stain in alcian blue solution for 30 minutes.
8. Rinse in distilled water.
9. Counterstain with nuclear fast red for 5 minutes.
10. Rinse in distilled water.
11. Dehydrate through graded alcohol and clear in xylene.
12. Coverslip using permanent mounting medium.

Results:

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

Strongly acidic sulfated mucosubstances --- blue

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

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

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, 2^nd Ed, 1980, pp 226-227, Battelle Press, Ohil.