48810 Kato Road, Suite 100E & 200E, Fremont, CA 94538 Emergo Europe, Prinsessegracht 20 2514 AP The Hague,

ht 20 2514 AP The Hague, The Netherlands

Tel : +1 (800) 421-4149, Fax: +1 (510) 824-1490
support@biogenex.com

SPECIAL STAINS
von Kossa Stain
Cat. No. SS045-50K
Doc. No. 932-SS045-50K Rev.No. E

Release Date: 30-Jun-2020

Intended Use
This product is intended for in vitro diagnostic use for demonstrating deposits of calcium in tissues.

Summary and Explanation

Abnormal deposits of calcium may be found in any area of the body. With the H&E stain, calcium appear deep blue-
purple.von Kossa staining is for demonstrating deposits of calcium or calcium salt and is not specific for the calcium ion
itself. When tissue sections are treated with silver nitrate solution the silver is deposited by replacing the calcium,
reduced by the strong light, and thereby visualized as metallic silver. Additional methods are sometimes employed along
with this technique to confirm the presence of calcium such as Alizarin Red, which detects calcium deposits.

Principles of the Procedure

The principle involved in von Kossa staining is a precipitation reaction in which silver ions react with phosphate (not
calcium) in the presence of acidic material. Photochemical degradation of silver phosphate to silver then occurs under
light illumination

Reagents Provided
Aqueous Silver Nitrate Solution (HK938- 14K) 1 x 13 ml, ready to use
Sodium Thiosulfate solution (HK905-14K) 1 x 13 ml, ready to use
Nuclear Fast Red Solution (HK895-14K) 1 x 13 ml, ready to use

Reagents Required but Not Supplied

Xylene
Mounting Medium
Positive Control Tissue (FS045)

Storage and Handling

Store all reagents at 2-8°C. Do not use after expiration dates as indicated on the reagent labels.

Specimen Preparation
Fixation plays an important role in preserving the tissue structures to be visualized using this stain. 10% neutral buffered
formalin is the preferred fixative; however, other fixatives may also be used. Ensure that the fixed sections are
adequately embedded in paraffin. Cut tissue sections to 4-5 microns.

von Kossa Manual Staining Protocol

1. Deparaffinize and hydrate the slides to running tap water for 2-3 minutes
2. Rinse the tissue sections in several changes of deionised water.
3. Add 250 µL of Aqueous silver nitrate solution on to the slide & incubate under U.V light for 20 minutes.
4. Rinse the tissue sections in several changes of deionised water.
5. Add 250 ul of Sodium thiosulfate & incubate for 5 minutes to remove un-reacted silver.
6. Rinse the tissue sections in several changes of deionised water.
7. Add 250ul of nuclear fast red & incubate for 5 minutes.
8. Rinse the tissue sections in deionised water for 4 times.

1
 48810 Kato Road, Suite 100E & 200E, Fremont, CA 94538 Emergo Europe, Prinsessegracht 20 2514 AP The Hague, The Netherlands
Tel : +1 (800) 421-4149, Fax: +1 (510) 824-1490
support@biogenex.com

9. Dehydrate through 95% alcohol and 2 changes of absolute alcohol, 3 minutes each.
10. Clear in Xylene and apply permanent mounting medium.

Quality Control
A positive control slide—one that will display positive staining with this stain—should be included in every test. This product
has been validated through extensive testing on the following tissue: calcium containing tissues and undecalcified bone.

Trouble shooting
1. Follow data sheet instructions correctly.
2. Gently invert all reagents prior to use.
3. The thickness of the section may affect the intensity of the staining. Necrotic tissue may exhibit non-specific staining.
4. Use tissue, which shows positive structure or element for which it is being tested.

Expected Results
Calcium salts appear black or brown black, Nuclei appear red & cytoplasm stains pink in colour.

Performance Characteristics
Specificity of Special Stain
von Kossa Stain is for the demonstration of calcium salts, in tissue sections.

BioGenex has conducted studies to evaluate the performance of all its Special Stains products. BioGenex Special Stains
have shown reproducible and consistent results when used with different lots. The products have been determined to be
stable for the periods specified on the labels either by standard real time or accelerated testing methods. BioGenex
ensures product quality through 100% quality control for all products released and through surveillance programs.

Bibliography

1. Sheehan D, Hrapchak B, Theory and Practice of Histotechnology, 2nd Ed, 1980, pp 226-227, Battelle Press,
Ohil.
2. Theory and Practice of Histotechnological Techniques, 4 th ed. JD Bancroft & A Stevens, eds., Churchill Livingstone,
New York (NY), 301-302, 1996.
3. Carson FL: Histotechnology: A self Instructional Text, ASCP Press, Chicago (IL), 194-196, 1990.
4. Clark G (1981) Staining Procedures. Williams and Wilkins, Baltimore, MD, pp 18