Reagents
- 2% HCL
- 2% Potassium ferrocyanide. < 2 grams of ferrocyanide to be dissolved in 100 ml of distilled water.
The working solution is to be made by pouring two stock solutions in equal volume. Should be prepared freshly before each staining procedure.
Staining Procedure
1) Deparaffinize the slide by heating in hot incubation.
2) To remove paraffin, the slide has to be dipped in xylene for 1-2 minutes. Repeat the process four times.
3) Keep the slide in alcohol for 1-2 minutes to remove xylene. Repeat the process two times.
4) Wash the slide in running water.
5) Pour the mixture over the slide and keep it for 30 minutes.
6) Wash in running water.
7) Cover the slide with 1% eosin and keep it for 30 seconds to 1 minute.
8) Wash the slide in running water.
9) Dry up, xylene, and mount.
Result
Hemosiderin is a golden brown granular pigment that is the product of the breakdown of hemoglobin and therefore occurs at the site of previous haemorrhage. The request for iron staining is, in reality, to check for the presence of hemosiderin in the section. However, hemosiderin should be differentiated from melanin, bile, and formalin pigments, which do not contain iron. The iron-containing pigment (hemosiderin) reacts with potassium ferrocyanide in an acid medium and yields a Prussian blue colour.
Note
Bone marrow slides should be washed in distilled water after fixation in methanol for at least 20 minutes before staining. Proceed further in the same way and use distilled water each time you wash the slide.