Hemosiderin Stain (Potassium Ferrocyanide)

 

 

Intended use:

Potassium ferrocyanide is intended for staining hemosiderin and differentiating hemosiderin from other pigments.

 

Principle:

The iron from tissue (in the ferric state) can be released from protein with dilute hydrochloric acid. It then reacts with potassium ferrocyanide to form blue ferric ferrocyanide, which is known as the Prussian blue reaction.

 

Methods:

1. Fix tissues in 10% neutral buffer formaldehyde. Dehydrate in typical manner and embed.

2. Deparaffinize tissue in distilled water. Rinse with distilled water for another time.

3. Mix an equal volume of 2% Potassium ferrocyanide and 2% hydrochloric acid. Drop the mixture onto slides for 20~30 minutes. Rinse with distilled water.

4. Counterstain in Nuclear Fast Red solution for 5~10 minutes and rinse briefly with distilled water.

5. Dehydrate in 95% ethanol and absolute ethanol, clear in xylene, and mount with mounting media.

 

Specifications:

 

Contents	3vialsx20ml/kit	3Btlsx100ml/kit	3Btlsx250ml/kit	Components
Potassium ferrocyanide solution	20ml	100ml	250ml	Potassium ferrocyanide
hydrochloric acid aqueous solution	20ml	100ml	250ml	Hydrochloric acid
Nuclear Fast Red solution	20ml	100ml	250ml	Nuclear Fast Red

 

Precaution:

1. Tissues should be fixed with neutral buffered formaldehyde. If ordinary formaldehyde is used, the tissue should be fixed for a shorter time before embedding. Tissues that are fixed in ordinary formaldehyde for a long time react poorly. Avoid using fixatives that contains chromate.

2. Hydrochloric acid of analytical grade or better is preferred as lower grade of hydrochloric acid typically contains iron impurities that can lead to false positive.

3. All containers/utensils used throughout the entire procedure should be clean. Avoid using any iron tools.

4. For steps prior to iron reaction, rinse only with distilled water to prevent iron in tap water from compounding with calcium salt in tissues that leads to false positive.

 

Expected Results:

 

Hemosiderin	——	Blue
Nuclei	——	Red