Von Kossa
Classification: pigment stain
Mechanism of staining: metallic substitution
Purpose: stain calcium
Positive controls: any calcified tissue, especially bone or teeth
Von Kossa
Placenta with minimal calcium shown as black precipitation
REAGENT |
PURPOSE |
MECHANISM OF STAINING |
SOURCE OF ERROR |
Silver nitrate |
Reacts with phosphate and carbonate groups |
Metallic Substitution Silver is substituted in place where calcium
was bound. Calcium in the body is mainly bound to phosphate and carbonate
radicals |
Omitted: No calcium
demonstrated. |
Too short: Minimal calcium or hard to see. |
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Too long: Non-specific staining possible. |
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UV Light or Hydroquinone |
Reducer |
Light or hydroquinone reduces the silver
salts to black metallic silver, macroscopically visible |
Omitted: Silver not detected. |
Too short: Minimal calcium may not be
demonstrated. |
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Too long: Precipitation. |
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5% Sodium Thiosulphate (optional): |
Fixative |
Remove excess silver and also fixative HYPO can prevent non-specific blackening but
may also render small amounts of calcium; also reduced silver can also be
partially-bleached with this step as well |
Omitted: Non-specific staining occurs. |
Too short: Few black precipitates might
occur. |
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Too long: No effect. |
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Neutral Red or van Gieson |
Counterstain |
Stains non-mineralized tissues, and RBCs |
Omitted: Tissue not containing calcium not demonstrated. |
Too short: Tissue component may be hard too
see. |
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Too long: Poor contrast with primary stain and small amounts
of calcium may be obscured. |
Von Koss - Positive
Placenta
Nuetral Red is weak and can be corrected by going back to alcohol and re-stain; gross calcium deposits.
Von Kossa - Negative
Placenta
Possibly no calcium present, acid fixative was used, no silver nitrate was added, there was no reduction or reduction time was too short
Special Considerations
Avoid acidic fixatives that dissolve calcium.
Rinse glassware before use with distilled water avoid contamination with free metal ions. Also use plastic forceps or wax coated forceps to handle slides.
References
Officer B. HIML 251 Lecture notes: Silver stains: Gordon and Sweet’s Reticulin and von Kossa, March 25, 2009