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Prussian blue staining (Perls’ Prussian blue) is a histochemical method that shows ferric iron (Fe3+) in tissue sections, most often the iron held in hemosiderin, as a blue deposit. It is qualitative, and it does not detect every form of iron.
Short answer
Prussian blue staining reveals ferric iron released from tissue iron stores. Ferric iron reacts with potassium ferrocyanide to form an insoluble blue compound, known as Prussian blue or Berlin blue, and nuclei are typically shown in red with a neutral red counterstain. The intensity of the blue signal indicates relative amount only: StainsFile describes the method as qualitative.
Definition and scope
The method is a special stain in histology, used alongside the routine and special stains described in our guide to histological staining. Its target is iron, and in practice that mostly means hemosiderin, the iron-storage pigment that pathologists look for in tissue sections. The 2025 chemistry review (Sonoda et al.) notes that most of the iron in tissues is present as ferric iron (FeIII), which is why the classic reaction works on so much tissue iron. For the wider context of how stained sections are read, see what is pathology.
How it works in practice
Two chemical steps explain every result. First, hydrochloric acid releases iron from hemosiderin as Fe3+. Second, the released ferric iron combines with potassium ferrocyanide to produce the blue compound. The result is ferric iron in blue and nuclei in red when neutral red is used as counterstain (StainsFile). This page deliberately omits reagent recipes and timings; follow your validated laboratory protocol and review the Safety Data Sheet of each chemical before handling it.
Prussian blue vs Turnbull blue
The two names are often confused. Perls’ reaction uses ferrocyanide to detect ferric iron. The Turnbull blue variant uses potassium ferricyanide, K3[FeIII(CN)6], to demonstrate ferrous iron (Fe2+). In the reaction chemistry reviewed by Sonoda and colleagues (2025), electron transfer from FeII to FeIII occurs immediately, so the end product has the same structure as Prussian blue. The two methods differ in which form of iron they start from, not in the colour you see.
What the stain can and cannot detect
| Iron form | Detected by Perls’ Prussian blue? | Source note |
|---|---|---|
| Hemosiderin iron (released as Fe3+ by HCl) | Yes | Sonoda et al. 2025; StainsFile |
| Ferrous iron (Fe2+) | Needs the Turnbull blue method | Sonoda et al. 2025 |
| Ferritin iron | Not microscopically, unless enhanced by DAB | Sonoda et al. 2025 |
| Haeme iron | No, because HCl does not release it | Sonoda et al. 2025 |
The same review states that Perls’ staining has an inherent limit of detection, so a negative section means no signal above that limit, not no iron.
Where it matters for labs and clinics: a pre-analytical checklist
Most failed or misleading iron stains trace back to handling, not to the staining step. Each point below is attributed to its source.
- Fixation: neutral-buffered formalin is suitable for fixing tissue (Sonoda et al. 2025). StainsFile warns that acid-containing fixatives may remove some iron deposits.
- Decalcification: solutions containing formic or nitric acid reduce the amount of hemosiderin (Sonoda et al. 2025).
- Iron contamination: avoid iron-containing materials and jars while fixing. Tap water and rust can cause false positives (StainsFile).
- Working solution: the staining solution should be prepared immediately before use (Sonoda et al. 2025).
- Rinsing: poor rinsing can leave a granular red deposit with a neutral red counterstain (StainsFile).
Slides and cover glass used in this work are covered in our guides to the microscope slide and the coverslip. Stored specimens are discussed under tissue bank.
Forensic use: a caution
Perls’ Prussian blue staining has also been applied to bruises in forensic work. The 2025 review cautions that bruises in infants and young children need particular care, and that reliability in intracranial and ocular regions is controversial.
Common misunderstandings
- “It measures how much iron is present.” It does not; intensity indicates relative amount and the method is qualitative only.
- “Prussian blue and Turnbull blue are different colours.” They give the same blue product from different starting forms of iron.
- “A blue-free section has no iron.” Haeme iron is not released by HCl and is unstainable, and ferritin iron is not seen microscopically without DAB enhancement.
- “Any acid-based workflow is fine.” Acid fixatives and some decalcifiers remove iron before staining.
FAQ
What does Prussian blue stain detect?
Ferric iron (Fe3+), mainly the iron in hemosiderin after HCl releases it, shown as blue.
Why is it called Prussian blue or Berlin blue?
These are two names for the same insoluble blue compound formed when ferric iron reacts with potassium ferrocyanide.
Is Prussian blue staining quantitative?
No. Staining intensity shows relative amount, but the method is qualitative only.
Can it detect ferrous iron?
Not with the standard Perls’ reaction; the Turnbull blue method, using potassium ferricyanide, is used for Fe2+.
Sources
- Sonoda A, Nihei M, Shinkawa N, Kakizaki E, Yukawa N. Perls’ Prussian blue staining and chemistry of Prussian blue and Turnbull blue. Forensic Sci Int Synerg. 2025;11:100627. pmc.ncbi.nlm.nih.gov/articles/PMC12275683 (checked 2026-10-29).
- StainsFile. Perls’ Prussian Blue for Hemosiderin. stainsfile.com (checked 2026-10-29).
