Von Kossa Stain - Calcium stain Flashcards
Classification:
Target tissue component:
Mechanism of staining:
Principle of staining:
Positive controls:
Classification: pigment stain
Target tissue component: calcium
Mechanism of staining: silver impregnation
Principle of staining: metallic substitution
Positive controls: any calcified tissue
What calcium look like in H&E
Purple blue
How is calcium demonstrated
von Kossa- metallic substitution reaction
AgNO3 reacts with non ionic radicals - PH4 and hco3 of carbon salts
Alizarin red S - dye lake reaction
What precautions should you take
Avoid Acid fixative - CA++ is soluble in mineral acids
Removes fixative pigments - to give non specific silver attachment
How is silver impregnation completed
5%Agno3 for 10-60 mins
-this forms silver phosphate and silver carbonates to where the silver used to be (metal substitution)
Expose slides to bright light to reduce silver salts to black metallic silver which is visible macroscopically
rinse in dH2O to slide off excess AgNO3
what is another method for reduction of silver nitrate
use a reducer photographic developer to see the silver reaction
-done in the dark
Hydroquinone - reducer
Sodium sulphite - stabilizer
dH2O - diluent
how to complete fixation
remove excess silver nitrate with hypo
to prevent non specific blackening
-even a small amount of CA causes false negative - OMIT if there is a small CA in tissue
wash well to remove hypo
what counter stain in used
red, van gieson or green counter stain
DCM
What will the results of this stain be
Mineralized bone and urates - dark brown to black
Non-mineralized tissue - as per counterstain
Nuclei - as per counterstain
Cytoplasm - as per counterstain
RBC - as per counterstain (yellow in van Gieson while the rest of tissues are red)
What is used as a Von Kossa control
Negative control for osteoid
Takes up counterstain
Negative control for urates
Urates turn black but soluble in LiCO3
Negative control for calcium
Treat slide with aqueous HCl for 5-10 min
What can remove the ca salts form tissue
Alcoholic iodine solution used to remove mercury pigment may also remove calcium salts from the tissues.
why do you need strong light
gives you black deposits
whereas little light gives brown deposits
