Ab485606

The kidney, liver, lung, and spleen were investigated to assess tissue microstructure, nanoparticle distribution, iron distribution, and oxidant injury after injection. Each organ was immersion fixed with 10% phosphate buffered formalin. Structure was assessed with hematoxylin and eosin (H&E). Iron was detected using Perls’ Prussian blue stain. Oxidative injury, macrophage infiltration, and ferritin distribution were assessed with immunohistochemistry (IHC). For IHC, tissue sections were submersed in H₂O₂ in methanol to quench endogenous peroxidase. Endogenous biotin was neutralized using an avidin-biotin (ABC) blocking kit (Vector Laboratories, Burlingame, CA). Oxidative stress was localized with 4-hydroxynonenal (4HNE) antibody (ab485606, Abcam, Cambridge, MA) at 1:2,000 dilution. Macrophages were identified using F4/80 antibody (ab6640, Abcam, Cambridge, MA). Ferritin was detected using immunofluorescence with antigen retrieval using 10mM citrate buffer, serum-free protein block (DAKO, Carpinteria, CA), anti-horse spleen ferritin primary antibody at 1:100 (F6136, Sigma Aldrich, St. Louis, MO), and donkey anti-rabbit secondary antibody at 1:500 (Life Technologies, Grand Island, NY). Sections were counterstained with 4′,6- Diamidino-2-Phenylindole, Dilactate (DAPI).

Tissue was harvested and fixed in 10% neutral buffered formalin, dehydrated and infiltrated with paraffin and sectioned at 4 μm thickness. General structure was demonstrated by staining with the Hematoxylin-Eosin procedure (HHS-128, Sigma, St. Louis MO). Picrosirius red staining was used to identify collagen deposition (Polysciences #09400, Warrington PA). For immunohistochemistry, sections were pretreated to quench endogenous peroxidase (H₂O₂ in methanol) and endogenous biotin (Avidin-Biotin blocking kit, Vector Laboratories, Burlingame, CA). Oxidative damage was assessed with antibodies against 4-hydroxynonenal (ab485606, Abcam, Cambridge MA, 1:2000 dilution) and 8 Hydroxyguanosine (ab48508, Abcam, Cambridge MA, 1:100 dilution)