Prussian blue reaction is a good method to distinctly exhibit ferric iron in tissues. Small amounts of ferric iron are found normally in spleen and bone marrow, present as ferroprotein and hemosiderin in mononuclear phagocyte or normoblast mitochondria. Any ferric ion in tissues can be differentiated out by hydrochloric acid, combines with the ferrocyanide under acid condition and results in the formation of a bright blue pigment called ferric ferrocyanide, or Prussian blue.
1.Deparaffinize and rehydrate sections to distilled water.
2.Prussian blue stain：Mix equal parts of hydrochloric acid and potassium ferrocyanide immediately before use. Immerse slides in this solution for 1 hour. Wash in distilled water, 2 changes.
3.Counterstain with nuclear fast red for 1-5 min, wash in running tap water.
4.Dehydrate through 3 changes of absolute alcohol, 5 min each. Clear in xylene, 2 changes and5 min each. Mount with resinous mounting medium.
5.Microscopy determine and analyze.
Iron (ferric form) is bright blue, and nuclei is red.
1. prussian blue stain working solution should be prepared according to the instructions immediately before use.
2. If precipitation occurs in Nuclear Fast Red Solution, filtrate it before use.
3. Use clear apparatus in the whole process, avoid metal utensils. Distilled water is better to use than tap water when washing slides and apparatus.
4. Do not use acid fix solution. Treat with chromate have adverse effect on preservation of potassium ferrocyanide.
5. Manage the staining time according to the ferric iron content.
6. Please pay attention to protect when operating.