Depex

All animals were perfused transcardially with 30 ml of saline, followed by 100 ml of 4% paraformaldehyde (diluted in 0.1 M PB, pH 7.4). Brains were removed from the skull and postfixed overnight at 4°C in the same solution. Subsequently, brains were cryoprotected by embedding in 30% sucrose in 0.1 M PB, at 4°C, for 48 h. In the BDA axonal labeling experiments, brains were freeze-sectioned in the coronal plane at 50 μm, and sections were collected in two parallel series. In the first series, after peroxidase activity blocking by incubation in H₂O₂ 0.66% (w/v) in 0.1 M PB for 15 min, sections were incubated for 2 h in avidin-biotin-peroxidase (1:100; Vectastain Elite, Vector Laboraries, Newark, CA, USA) diluted in 0.1 M PB. After washing, peroxidase was visualized using the glucose oxidase-3-3′diaminobenzidine (Sigma-Aldrich Química, Madrid, Spain) nickel sulfate-enhanced method (Shu et al., 1988 (link)). For labeling localization, the same sections were then counterstained using cytochrome C-oxidase (CyO) histochemistry (Wong-Riley et al., 1978 (link)) and finally mounted and coverslipped with DePeX (Serva Electrophoresis GMbH, Heidelberg, Germany). A second series was kept in antifreeze solution at −20°C as a backup.

The immunohistochemical procedures are described in detail in Protocol S9. Briefly, specimens were deparaffinized in xylene and hydrated. Then, antigen retrieval was performed with a microwave step and the corresponding buffer, as listed in Table 2. Afterwards, endogenous peroxidases were inhibited with 3 % H₂O₂ (Merck, Darmstadt, Germany) followed by the blocking of non-specific binding sites with 5 % normal goat serum (Vector Laboratories, Newark, CA, USA). According to Table 2, primary antibodies were diluted and incubated overnight at 4 °C. Subsequently, specimens were incubated with secondary antibodies (see Table 2) and ABC-HRP kit (Vectastain Elite, Newark, CA, USA). Visualization was performed by adding 3,3′-diaminobenzidine–tetrahydrochloride (DAB; Sigma Aldrich, Merck, Darmstadt, Germany) for 7 min. After stopping the reaction in tap water, counterstaining with hematoxylin was performed for less than 10 s. Specimens were then dehydrated, mounted in DePeX (Serva, Heidelberg, Germany), and coverslipped. Negative controls were performed for each reaction while omitting the primary antibody.

Liver samples were fixed in 4% buffered formalin and embedded in paraffin. Deparaffinized tissue sections were stained with H&E using standard procedures and images were obtained using a high-resolution Leica DC200 digital camera mounted on an Olympus DMLB microscope. Immunohistochemistry of the macrophage marker F4/80, a kind gift of Dr. Antonio Castrillo, was performed on 4 μm deparaffinized-rehydrated sections with 1:100 antibody dilution. Antigen retrieval was carried out with citrate buffer and endogenous peroxidase activity was inhibited with 3% H₂O₂. Samples were blocked and incubated overnight with the antibody, and signal was amplified with the ABC Kit (Vectastin). Slides were revealed with DAB (Vector), counterstained with Hematoxylin and mounted with DePeX (Serva).

The rehydrated paraffin sections were incubated in a solution of 5-bromo-4-chloro-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT, KPL, Gaithersburg, MD, USA) salt for 45 min in a moist chamber at 37°C. After thoroughly washing in aqua dest., sections were counterstained with nuclear fast red, dehydrated, and coverslipped with DePex (Serva, Heidelberg, Germany).

After the internalization of IONPs in U251 cells or hMSC by DI or CMI, the cells were washed twice with PBS (AMRESCO, Ohio, USA) and fixed with 4% paraformaldehyde solution for 30 minutes at room temperature. Again, cells were washed twice with PBS, and then were incubated with a 1:1 mixture of 4% potassium ferrocyanide (Sigma-Aldrich) and 4% hydrochloric acid (Sigma-Aldrich) (Prussian blue staining solution) for 15 minutes at room temperature and washed with distilled water three times. The counterstaining was done for cytoplasm with neutral red 0.5% (Panreac Química S.L.U) for 2 minutes at room temperature. After drying the cells, a cover slip was mounted by using the mounting medium DePeX (SERVA Electrophoresis GmbH) and finally, the cells were observed using light microscopy (Leica DMI3000B, Leica Microsystems, Germany). All experiments were carried out in triplicate. A table with the total number of cells considered for all experiments are available in Supplementary Section 2 (Suppl. Table S4).