Aim: To demonstrate the presence of iron in tissues.

Principle: The ferric iron in tissue combines with potassium ferro cyanide to form ferric-ferro cyanide. This compound has bright blue color (prussian blue). Prussian blue precipitate is insoluble, hence it can be combined with other staining methods.

Control: Hemosiderin positive tissue

Reagents:-

1. 2%Hydrochloric acid

concentrated hydrochloric acid               2ml
Distilled water                                            98ml

2. 2% potassium ferrocyanide

Potassium ferrocyanide                         2mg
Distilled water                                         100ml

3. 0.15% Basic fuchsin

Basic fuchsin                                           0.15g
50% ethyl alcohol                                   100ml

Procedure:-

1. Bring section to water.
2. Mix equal volume of 2% potassium ferrocyanide and 2% hydrochloric acid.
Pour the solution on the slide and keep it for 20 minutes.
3. Wash thoroughly with water.
4. Counter-stain with basic fuchsin or eosin for 30 seconds.
5. Wash with water, dehydrate, clear in xylene and mount in DPX.

Result:-

Ferric iron                            blue 
Nuclei                                  red 
Other tissues                     shades of pink

Note: All traces of ferrocyanide should be removed before it is counter-stained,
otherwise a dark red fine precipitate will form.