Skip to main content
SpringerLink
Log in

Prussian Blue Staining to Visualize Iron Oxide Nanoparticles

  • Protocol
  • First Online:
Histochemistry of Single Molecules

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2566))

Abstract

Iron deposits in cells and tissues can be detected by ex vivo histological examination through the Prussian blue (PB) staining. This practical, inexpensive, and highly sensitive technique involves the treatment of fixed tissue sections and cells with acid solutions of ferrocyanides that combine with ferric ion forming a bright blue pigment (i.e., ferric ferrocyanide). The staining can be applied to visualize iron oxide nanoparticles (IONPs), versatile magnetic nanosystems that are used in various biomedical applications and whose localization is usually required at a higher resolution than that enabled by in vivo tracking techniques.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Pereira C, Pereira AM, Fernandes C, Rocha M, Mendes R, Paz Fernández-García M et al (2012) Superparamagnetic MFe2O4 (M = Fe, Co, Mn) nanoparticles: tuning the particle size and magnetic properties through a novel one-step coprecipitation route. Chem Mat 24:1496–1504

    Article  CAS  Google Scholar 

  2. Wu W, Wu Z, Yu T, Jiang C, Kim WS (2015) Recent progress on magnetic iron oxide nanoparticles: synthesis, surface functional strategies and biomedical applications. Sci Technol Adv Mater 16:023501

    Article  Google Scholar 

  3. Stueber DD, Villanova J, Aponte I, Xiao Z, Colvin VL (2021) Magnetic nanoparticles in biology and medicine: past, present, and future trends. Pharmaceutics 13:943

    Article  CAS  Google Scholar 

  4. Filippi M, Nguyen DV, Garello F, Perton F, Bégin-Colin S, Felder-Flesch D et al (2019) Metronidazole-functionalized iron oxide nanoparticles for molecular detection of hypoxic tissues. Nanoscale 11:22559–22574

    Article  CAS  Google Scholar 

  5. Filippi M, Born G, Felder-Flesch D, Scherberich A (2020) Use of nanoparticles in skeletal tissue regeneration and engineering. Histol Histopathol 35:331–350

    CAS  PubMed  Google Scholar 

  6. Filippi M, Dasen B, Guerrero J, Garello F, Isu G, Born G et al (2019) Magnetic nanocomposite hydrogels and static magnetic field stimulate the osteoblastic and vasculogenic profile of adipose-derived cells. Biomaterials 223:119468

    Article  CAS  Google Scholar 

  7. Xia Y, Sun J, Zhao L, Zhang F, Liang XJ, Guo Y et al (2018) Magnetic field and nano-scaffolds with stem cells to enhance bone regeneration. Biomaterials 183:151–170

    Article  CAS  Google Scholar 

  8. Liu XL, Chen S, Zhang H, Zhou J, Fan HM, Liang XJ (2019) Magnetic nanomaterials for advanced regenerative medicine: the promise and challenges. Adv Mater 31:e1804922

    Article  Google Scholar 

  9. Li Y, Ye D, Li M, Ma M, Gu N (2018) Adaptive materials based on iron oxide nanoparticles for bone regeneration. ChemPhysChem 19:1965–1979

    Article  CAS  Google Scholar 

  10. Park J, Jin C, Lee S, Kim JY, Choi H (2019) Magnetically actuated degradable microrobots for actively controlled drug release and hyperthermia therapy. Adv Healthc Mater 8:e1900213

    Article  Google Scholar 

  11. Choi J, Hwang J, Kim JY, Choi H (2021) Recent progress in magnetically actuated microrobots for targeted delivery of therapeutic agents. Adv Healthc Mater 10:e2001596

    Article  Google Scholar 

  12. Peyer KE, Zhang L, Nelson BJ (2013) Bio-inspired magnetic swimming microrobots for biomedical applications. Nanoscale 5:1259–1272

    Article  CAS  Google Scholar 

  13. Kievit FM, Zhang M (2011) Surface engineering of iron oxide nanoparticles for targeted cancer therapy. Acc Chem Res 44:853–862

    Article  CAS  Google Scholar 

  14. Wu W, He Q, Jiang C (2008) Magnetic iron oxide nanoparticles: synthesis and surface functionalization strategies. Nanoscale Res Lett 3:397–415

    Article  CAS  Google Scholar 

  15. Bitonto V, Alberti D, Ruiu R, Aime S, Geninatti Crich S, Cutrin JC (2020) L-ferritin: a theranostic agent of natural origin for MRI visualization and treatment of breast cancer. J Control Release 319:300–310

    Article  CAS  Google Scholar 

  16. Baroni S, Ruggiero MR, Bitonto V, Broche LM, Lurie DJ, Aime S et al (2020) In vivo assessment of tumour associated macrophages in murine melanoma obtained by low-field relaxometry in the presence of iron oxide particles. Biomaterials 236:119805

    Article  CAS  Google Scholar 

  17. Bulte JW (2009) In vivo MRI cell tracking: clinical studies. Am J Roentgenol 193:314–325

    Article  Google Scholar 

  18. Svenskaya Y, Garello F, Lengert E, Kozlova A, Verkhovskii R, Bitonto V et al (2021) Biodegradable polyelectrolyte/magnetite capsules for MR imaging and magnetic targeting of tumors. Nano 5:362–377

    Google Scholar 

  19. Filippi M, Dasen B, Scherberich A (2021) Rapid magneto-sonoporation of adipose-derived cells. Materials (Basel) 14:4877

    Article  CAS  Google Scholar 

  20. Wierzbinski KR, Szymanski T, Rozwadowska N, Rybka JD, Zimna A, Zalewski T et al (2018) Potential use of superparamagnetic iron oxide nanoparticles for in vitro and in vivo bioimaging of human myoblasts. Sci Rep 8:3682

    Article  Google Scholar 

  21. Schlorf T, Meincke M, Kossel E, Glüer CC, Jansen O, Mentlein R (2010) Biological properties of iron oxide nanoparticles for cellular and molecular magnetic resonance imaging. Int J Mol Sci 12:12–23

    Article  Google Scholar 

  22. Ma W, Gehret PM, Hoff RE, Kelly LP, Suh WH (2019) The investigation into the toxic potential of iron oxide nanoparticles utilizing rat pheochromocytoma and human neural stem cells. Nano 9:453

    CAS  Google Scholar 

  23. Boutry S, Forge D, Burtea C, Mahieu I, Murariu O, Laurent S et al (2009) How to quantify iron in an aqueous or biological matrix: a technical note. Contrast Media Mol Imaging 4:299–304

    Article  CAS  Google Scholar 

  24. Churukian CJ (2008) Pigments and minerals. In: Bancroft JD, Gamble M (eds) Theory and practice of histological techniques, 6th edn. Churchill Livingstone

    Google Scholar 

  25. Vasanawala SS, Nguyen KL, Hope MD, Bridges MD, Hope TA, Reeder SB et al (2016) Safety and technique of ferumoxytol administration for MRI. Magn Reson Med 75:2107–2111

    Article  Google Scholar 

  26. Meguro R, Asano Y, Odagiri S, Li C, Iwatsuki H, Shoumura K (2007) Nonheme-iron histochemistry for light and electron microscopy: a historical, theoretical and technical review. Arch Histol Cytol 70:1–19

    Article  CAS  Google Scholar 

  27. Schroeter M, Saleh A, Wiedermann D, Hoehn M, Jander S (2004) Histochemical detection of ultrasmall superparamagnetic iron (USPIO) contrast medium uptake in experimental brain ischemia. Magn Reson Med 52:403–406

    Article  Google Scholar 

  28. Nguyen-Legros J, Bizot J, Bolesse M, Pulicani JP (1980) “Diaminobenzidine black” as a new histochemical demonstration of exogenous iron. Histochemistry 66:239–244

    Article  CAS  Google Scholar 

  29. Roschzttardtz H, Conéjéro G, Curie C, Mari S (2010) Straightforward histochemical staining of Fe by the adaptation of an old-school technique: identification of the endodermal vacuole as the site of Fe storage in Arabidopsis embryos. Plant Signal Behav 5:56–57

    Article  CAS  Google Scholar 

  30. van Duijn S, Nabuurs RJ, van Duinen SG, Natté R (2013) Comparison of histological techniques to visualize iron in paraffin-embedded brain tissue of patients with Alzheimer’s disease. J Histochem Cytochem 61:785–792

    Article  Google Scholar 

  31. Sands SA, Leung-Toung R, Wang Y, Connelly J, LeVine SM (2016) Enhanced histochemical detection of iron in paraffin sections of mouse central nervous system tissue: application in the APP/PS1 mouse model of Alzheimer's disease. ASN Neuro 8:1759091416670978

    Article  Google Scholar 

  32. Moos T, Mollgard K (1993) A sensitive post-DAB enhancement technique for demonstration of iron in the central nervous system. Histochemistry 99:471–475

    Article  CAS  Google Scholar 

  33. Mieloch AA, Żurawek M, Giersig M, Rozwadowska N, Rybka JD (2010) Bioevaluation of superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with dihexadecyl phosphate (DHP). Sci Rep 10:2725

    Article  Google Scholar 

  34. Frank JA, Kalish H, Jordan EK, Anderson SA, Pawelczyk E, Arbab AS (2007) Color transformation and fluorescence of Prussian blue-positive cells: implications for histologic verification of cells labeled with superparamagnetic iron oxide nanoparticles. Mol Imaging 6:212–218

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Molecular Biotechnology and Health Sciences, University of Turin, Torino, Italy

    Valeria Bitonto & Francesca Garello

  2. Department of Biomedicine, University and University Hospital of Basel, Basel, Switzerland

    Arnaud Scherberich

  3. Department of Biomedical Engineering, University of Basel, Allschwil, Switzerland

    Arnaud Scherberich

  4. Soft Robotics Laboratory, ETH Zurich, Zurich, Switzerland

    Miriam Filippi

Authors
  1. Valeria Bitonto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Francesca Garello
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Arnaud Scherberich
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Miriam Filippi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Arnaud Scherberich or Miriam Filippi .

Editor information

Editors and Affiliations

  1. Department of Biology and Biotechnology, University of Pavia, PAVIA, Italy

    Carlo Pellicciari

  2. Department of Biology and Biotechnology, University of Pavia, PAVIA, Italy

    Marco Biggiogera

  3. Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, VERONA, Italy

    Manuela Malatesta

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Bitonto, V., Garello, F., Scherberich, A., Filippi, M. (2023). Prussian Blue Staining to Visualize Iron Oxide Nanoparticles. In: Pellicciari, C., Biggiogera, M., Malatesta, M. (eds) Histochemistry of Single Molecules. Methods in Molecular Biology, vol 2566. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2675-7_26

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2675-7_26

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2674-0

  • Online ISBN: 978-1-0716-2675-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation