Prussian blue stain
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Small amounts of ferric iron are found normally in the spleen and bone marrow. Excessive amounts are present in hemochromatosis and hemosiderosis. Prussian blue reaction involves the treatment of sections with acid solutions of ferrocyanides. Any ferric ion (+3) present in the tissue combines with the ferrocyanide and results in the formation of a bright blue pigment called Prussian blue, or ferric ferrocyanide. This is one of the most sensitive histochemical tests and will demonstrate even single granules of iron in blood cells.
Fixation: 10% Formalin.
Sections: Paraffin sections at 5 um.
1) Deparaffinize and hydrate sections to distilled water.
2) Mix equal parts of hydrochloric acid and potassium ferrocyanide prepared immediately before use. Immerse slides in this solution for 20 minutes.
3) Wash in distilled water, 3 changes.
4) Counterstain with nuclear fast red for 5 minutes.
5) Rinse twice in distilled water.
6) Dehydrate through 95% and 2 changes of 100% alcohol.
7) Clear in xylene, 2 changes, 3 minutes each.
8) Coverslip with resinous mounting medium.
Iron (ferric form): bright blue