title: Combined in situ zymography, immunofluorescence, and staining of iron oxide particles in paraffin-embedded, zinc-fixed tissue sections.
abstract: Superparamagnetic iron oxide particles are used as potent contrast agents in magnetic resonance imaging. In histology, these particles are frequently visualized by Prussian blue iron staining of aldehyde-fixed, paraffin-embedded tissues. Recently, zinc salt-based fixative was shown to preserve enzyme activity in paraffin-embedded tissues. In this study, we demonstrate that zinc fixation allows combining in situ zymography with fluorescence immunohistochemistry (IHC) and iron staining for advanced biologic investigation of iron oxide particle accumulation. Very small iron oxide particles, developed for magnetic resonance angiography, were applied intravenously to BALB/c nude mice. After 3 hours, spleens were explanted and subjected to zinc fixation and paraffin embedding. Cut tissue sections were further processed to in situ zymography, IHC, and Prussian blue staining procedures. The combination of in situ zymography as well as IHC with subsequent Prussian blue iron staining on zinc-fixed paraffin-embedded tissues resulted in excellent histologic images of enzyme activity, protease distribution, and iron oxide particle accumulation. The combination of all three stains on a single section allowed direct comparison with only moderate degradation of fluorescein isothiocyanate-labeled substrate. This protocol is useful for investigating the biologic environment of accumulating iron oxide particles, with excellent preservation of morphology.
authors.affiliation: Charté-Radiology, Charitéplatz 1 Berlin, Berlin 10117, Germany. eyk.schellenberger@charite.de
authors.forename: Akvile
authors.initials: A
authors.lastname: Haeckel
authors.forename: Lena
authors.initials: L
authors.lastname: Schoenzart
authors.forename: Franziska
authors.initials: F
authors.lastname: Appler
authors.forename: Joerg
authors.initials: J
authors.lastname: Schnorr
authors.forename: Matthias
authors.initials: M
authors.lastname: Taupitz
authors.forename: Bernd
authors.initials: B
authors.lastname: Hamm
authors.forename: Eyk
authors.initials: E
authors.lastname: Schellenberger
mesh_terms: D000818:Animals; D004396:Coloring Agents; D005295:Ferrocyanides; D005404:Fixatives / Q000737:chemistry; D007150:Immunohistochemistry / Q000379:methods*; D007501:Iron / Q000032:analysis; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D020778:Matrix Metalloproteinase 2 / Q000032:analysis; D020780:Matrix Metalloproteinase 9 / Q000032:analysis; D051379:Mice; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D016612:Paraffin Embedding; D013154:Spleen / Q000737:chemistry / Q000378:metabolism; D016707:Tissue Fixation / Q000379:methods*; D015032:Zinc / Q000737:chemistry*
chemical_list: D004396:Coloring Agents; D005295:Ferrocyanides; D005404:Fixatives; D058185:Magnetite Nanoparticles; D007501:Iron; D020778:Matrix Metalloproteinase 2; C522362:Mmp2 protein, mouse; D020780:Matrix Metalloproteinase 9; C501830:Mmp9 protein, mouse; D015032:Zinc; C000170:ferric ferrocyanide