Prussian Blue
a Histological Stain Used to Detect Iron Deposits in Tissues, Highlighting Ferric Iron .
Author: Kelsey Goldman, HTL (ASCP)
Last Update: 24 March 2025
Copyright: 2025, HistologyOutlines.com
Cite this Page! -> Goldman, Kelsey. “Prussian Blue.” HistologyOutlines.Com, 24 March 2025. Accessed (today).
Purpose
- Prussian blue is a stain that detects ferric iron. Ferric iron is found naturally in some tissues, such as the bone marrow. However, in the case of rare blood conditions it may be found in aggregates in other tissues. Ferric iron refers to Fe3+, this means that there are three iron atoms ionically joined together. This is an important differentiation from ferrous iron, which is Fe2+ (Carson, 2001).
Control Tissues
- Tissue containing moderate amounts to ferric iron, too much may contaminate other samples in the solution.
Staining Pattern
- Nuclei - Red
- Hemosiderin (iron, Fe3+) - Bright Blue
- Other Tissue Elements - Pink
Biochemistry Theory
- When the ferric iron is stained with potassium ferrocyanide in an acidic environment the iron forms insoluble Prussian blue pigment (Carson, 2001).
- There are a few different configurations of iron that can be found in tissue. It is important when staining to be detecting a compound as specifically as possible. Prussian Blue is for the detection of Ferric iron (Fe 3+). The Potassium ferrocyanide of the Prussian blue binds with the salts of ferric iron to form an insoluble brilliant blue pigment (Disbrey, 1970).
Troubleshooting
- Make sure coplin jars are clean. If they had been previously used to stain slides with iron hematoxylin or ferric chloride solutions, then contaminated staining will occur (Carson, 2001).
- To avoid contamination, use clean plastic forceps. Using metal forceps could introduce contamination from any iron present in the metal (Carson, 2001).
- Do not decal samples that will be stained for iron. The iron may be dissolved in the decalcification process (Carson, 2001).
- Nuclear fast red ( the preferred counterstain) will create a cloudy residue on the slide if excess is not properly washed off (Carson, 2001).
- 10% NBF or alcohol can be used for fixatives
Additional References
- Carson, F. L. (2001). Histotechnology: a self-instructional text (2. ed., [Reprint]). ASCP Press.
- Disbrey, B. (1970). Histological Laboratory Methods. E & S Livingstone.
Sample Protocol
Protocol: Prussian Blue
Adapted from Carson's
Reagents:
- Potassium Ferrocyanide 2%
- Hydrochloric Acid 2%
- Nuclear Fast Red
Method:
- Deparaffinize and rehydrate. From this point forward, use care to not introduce contaminant iron (see troubleshooting).
- Create a 1:1 solution of Potassium ferrocyanide and Hydrochloric Acid. Place the slides in the solution and heat the solution for 20 minutes at 60°C.
- Wash in several changes of distilled water.
- Counterstain in nuclear fast red for 5 minutes.
- Wash in running tap water for a minimum of 1 minute.
- Dehydrate in graded alcohols and bring to xylene. Coverslip and mount in a xylene-based mounting media.