Von Kossa Staining Protocol for Calcium

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

Von Kossa Stain For Calcium

In histology, the von kossa stain for calcium is commonly used to detect abnormal calcium deposits in the body. The transformation of calcium salts into silver salts is based on the replacement of calcium ions bound to phosphates by silver ions brought in by a solution of silver nitrate.

When silver phosphates are exposed to light, they undergo photochemical breakdown, resulting in the formation of metallic silver deposits. Nuclear red counterstaining permits the nuclei of the cells to be stained in red while the cytoplasm is stained in pink.

Von kossa stain for calcium can be used to diagnose calcinosis (eg: chondrocalcinosis, nephrocalcinosis, cutaneous calcinosis). It can also be utilised to find tumour calcifications. Von kossa stain for calcium can be used on any tissue that has calcium deposits that are abnormal. Joints, blood vessels, and kidneys are the most commonly affected organs.

Principle

Silver ions are displaced from solution by carbonate or phosphate ions, due to their respective positions in the electrochemical series.  The argentaffin reaction is photochemical in nature, and the activation 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.

Requirements

Chemicals

1. Silver Nitrate

2. Sodium Thiosulphate

3. Neutral Red

4. Glacial Acetic Acid

Equipment’s

1. UV light

2. Light Microscope

Reagents

1. 1% Aqueous Silver Nitrate Solution: 5gm of Silver nitrate dissolved in 50 ml Distilled water and make up to 100 ml for Von kossa stain for calcium.

2. 5% Sodium Thiosulfate: 2 gm of Sodium thiosulfate dissolved in 50 ml Distilled water and make up to 100 ml.

3. Dissolve the 1.0 g of Neutral red in the 100.0 ml double distilled water.  Add the Glacial acetic acid and Mix well for Von kossa stain for calcium use.

Procedure

1. Deparaffinize and hydrate paraffin sections in double distilled water. Rinse with distilled water several times.

2. Flood slides with 5% aqueous silver nitrate, Expose sections to bright sunlight, or UV lamp 20 mins.

3. Wash well in distilled water and Treat sections with 2% sodium thiosulphate for approximately 2 mins.

4. Wash the slides running tap water carefully and wash the slides again with double distilled water.

5. Counterstain with 1% Neutral red 2 mins and rapidly dehydrate, clear and permanent mounting medium such as DPX is used for the coverslip.

Results

Von Kossa Staining

Calcium sites: Black

Collagen: Red

Osteoid: Red

Further Readings

Reference

  1. https://www.abcam.com/von-kossa-stain-kit-calcium-stain-ab150687.html
  2. https://pubmed.ncbi.nlm.nih.gov/12724828/