⑫ Staining

Kossa silver impregnation method

Calcium salt is stained a deep brown black color.

Staining procedure

1	Deparaffinization	Xylene	3 changes, 10 minuntes each
2	Removal of xylene	100% ethanol	3 changes, 5 minutes each
3	Hydration	95%, 70% ethanol	5 minutes each
4	Washing	Running tap water	2 minutes
5	Rinsing	Distilled water
6	Silver impregnation	5% silver nitrate solution	Under indirect light for 10 minutes to 1 hour
7	Washing	Distilled water	Several changes
8	Fixing	5% sodium thiosulfate solution	1 minute
9	Washing	Running tap water	10 minutes
10	Counterstaining	Kernechtrot	5 minutes
11	Washing	Running tap water	10 minutes
12	Dehydration	70% , 95% ethanol	5 minutes each
13	Dehydration	100% ehtanol	3 changes, 5 minutes each
14	Clearing	Xylene	3 changes, 10 minuntes each
15	Coverslipping

Staining method

Deparaffinization
Wash in distilled water
Incubate in 5% silver nitrate under indirect light for 10 minutes to 1 hour
Place in 5% sodium thiosulfate for 1 minute
Flushing
Nuclei are stained red with Kernechtrot.
Flushing
Dehydration, penetration, and encapsulation

Staining results

Areas of calcification are stained black.

Calcium phosphate is mainly targeted.

Please be aware that copper, mercury, and lead also react with this method.