STANDARD OPERATING PROCEDURE FOR VON KOSSA STAIN

Von Kossa Stain

Principle: Von Kossa stain is to detect the presence of abnormal calcium deposits present in the tissue. Tissue sections are treated with silver nitrate solution, and the calcium is reduced by the strong light and replaced with silver deposits, visualized as metallic silver.

Control: Un decalcified bone

Fixative: Formalin fixed paraffin embedded tissue section

Section: 3-5micron section

Reagents

1. 5% aqueous silver nitrate solution

2. 2-5% aqueous sodium thiosulfate

3. 1% Neutral red/Nuclear Fast Red

4. Neutral red- 1.0g

5. Distill water- 100ml

6. Glacial Acetic Acid- 1ml

Dissolve the dye in the distilled water. Add the acid. Mix well. Filter the reagent before use


Procedure

1. Deparaffinize and hydrate to distilled water.

2. Flood slides with 5% Silver solution

3. Place the slides in bright sunlight, or in front of a 60-watt lamp, and place foil (or mirror) behind the jar to reflect the light. Leave for 1 hour or until calcium turns black.

3. Rinse in distilled water, 3 changes.

4. Treat sections with 2-5% sodium thiosulphate for 2-5 minutes.

5. Wash in running tap water, and rinse in distilled water.

6. Counterstain with 1% Neutral Red/ Nuclear fast red for 2-5 minutes.

7. Wash in tap/distilled water.

8- Dehydrate, clear, and mount the stained slides.


Result:

Calcium sites: Black

Collagen: Red

                                                                    END OF THE DOCUMENT

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