Von Kossa Staining Protocol for Bone

Von Kossa staining protocol for bone post we have briefly explained about Von Kossa stain method’s principle, requirements, von kossa stain for calcium procedure, result with Label in von kossa stain for calcium.

Von Kossa Stain For Calcium

Von kossa stain for calcium is used to demonstrate calcium deposits or calcium salts and is not specific to the calcium ion itself. Tissue sections are treated with a silver nitrate solution in this method, and the silver is deposited by replacing the calcium reduced by the strong light, and thus visualised as metallic silver.

Acid mucosubstances and acetic mucins are stained by alcian blue. Excessive amounts of non-sulfated acidic mucosubstances are found in mesotheliomas; normal amounts occur in blood vessel walls but increase in early atherosclerosis lesions. Mucosubstances that are highly acidic will be stained blue, nuclei will be stained pink to red, and cytoplasm will be pale pink.

When von Kossa and Alcian Blue staining are used together, both calcium and mucosubstances are visible on the same tissue sections in von kossa stain for calcium.

Principle

Tissue sections are treated with a silver nitrate solution; the strong light reduces the calcium and replaces it with silver deposits, which are visible as metallic silver in von kossa stain for calcium.

Requirements

Equipment’s

1. Acid cleaned glassware

2. 60-watt lamp

3. Mirror

Chemicals

1. Silver nitrate

2. Sodium thiosulfate

3. Alcian blue

4. Acetic acid

5. Nuclear fast red

6. Aluminum sulfate

Controls

Positive Control: 16-18 days mouse embryo and Tissue containing known positive calcium deposits, or undecalcified bone in von kossa stain for calcium

Fixation: Formalin fixed, paraffin embedded tissue sections or alcohol fixed, frozen (Cryotome) sections.

Reagents

1. Silver Nitrate Solution

1% Aqueous Silver Nitrate Solution: 1 gm of Silver nitrate dissolved in 50 ml Distilled water and make upto 100 ml. 

2. 5% Sodium Thiosulfate

5% Sodium Thiosulfate: 5 gm of Sodium thiosulfate dissolved in 50 ml Distilled water and make upto 100 ml. 

3. Alcian Blue Solution

1 gm of Alcian blue, 8GX dissolved in 50 ml Distilled water, Mix well and adjust pH to 2.5 using acetic acid and make upto 100 ml. 

4. 0.1% Nuclear Fast Red

0.1 gm of nuclear fast red and 5 g Aluminum sulfate dissolved in 100 ml Distilled water. 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 and hydrate paraffin sections in double distilled water. Rinse with distilled water several times for von kossa stain for calcium procedure.

2. In a clear glass copulin jar placed under ultraviolet light, incubate sections for 20 minutes in a 1 percent silver nitrate solution (or in front of a 60-100 watt light bulb for 1 hour or longer).

Note: If the von kossa stain for calcium was weak or rinsed away during the washing process, it indicated that the UV light was not strong enough. A longer staining time of up to several hours is required.

3. Rinse with distilled water several times. Remove unreacted silver by soaking it in 5 percent sodium thiosulfate for 5 minutes and then rinsing it in distilled water.

4. Rinse in distilled water after staining in alcian blue solution for 30 minutes. For 5 minutes, counterstain with nuclear fast red. Rinse thoroughly with distilled water.

5. Dehydrate in graded alcohol, and then clear in xylene. Permanent mounting medium such as DPX is used for the coverslip for further von kossa stain for calcium.

Results

Von Kossa Staining

Von Kossa Stain For Calcium

Calcium salts: Black or brown-black

Strongly acidic sulfated mucosubstances:  Blue

Nuclei: Red

Cytoplasm: Pink

Further Readings

Reference