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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
Procedure:
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.
Results:
Iron (ferric
form) ------------------------ bright blue
Nuclei
------------------------------------- red
Cytoplasm -------------------------------- pink
References:
Theory and Practice of Histotechnology