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Prussian Blue Staining Protocol for Iron
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Prussian Blue Staining Protocol for Iron

Description: 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.

Solutions and Reagents:

20% Aqueous Solution of Hydrochloric Acid:

      Hydrochloric acid, concentrated ------------ 20 ml

      Distilled water -------------------------------- 80 ml

      Mix well.

10% Aqueous Solution of Potassium Ferrocyanide:

      Potassium ferrocyanide, Trihydrate

      (K4Fe(CN)6.3H2O, FW 422.4, Sigma, Cat# P-3289) -----10 g

      Distilled water ----------------------------------------------- 100 ml

      Mix to dissolve

Working Solution: Mix equal parts of 20% hydrochloric acid and 10% potassium ferrocyanide solution JUST before use.

Nuclear Fast Red Solution


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

      Nuclei ------------------------------------- red

      Cytoplasm -------------------------------- pink


      Theory and Practice of Histotechnology

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