Prussian blue

Five min, 30 min, 1 h, 6 h, 1 day, 3 days, 5 days, 10 days and 20 days after ocular microinjection, Xenopus embryos were formalin fixed for 1 h, after which they were embedded in paraffin and sectioned (14 μm). Detachment of the neuroretina from RPE was found on the control and injected eye to the same degree, and was consistent with tissue processing artefacts [38 (link)].
One day after ocular microinjection, zebrafish embryos were fixed in 4% paraformaldehyde for 2 h, after which they were embedded in paraffin and sectioned (10 μm). For cryostat sections the embryos were fixed in 4% paraformaldehyde in PBS for 2 h and cryoprotected with 20% sucrose in PBS for 2 h, then they were embedded in OCT cryostat embedding medium Tissue Tek^® (Sakura, Finetek, Torrance, CA, USA) and criosectioned (12 μm). Nuclei staining was performed using hoechst 33342.
The cryo- and paraffin sections were stained by Prussian Blue according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA) after a treatment of pigment bleaching in 50% formamide-1% hydrogen peroxide in presence of cold light.

Sudan Black, Prussian Blue, ( ±) α-LA, Luperox® DI (tert-Butyl peroxide), anti-fatty acid synthase (FAS), and trypsin were purchased from Sigma Chemical Co. (St. Louis, MO). BODIPY® 598/591 C11, MitoTracker Deep Red FM, DAPI, were purchased from Invitrogen/Molecular Probes (Eugene, OR). MitoPeDPP® was purchased from Dojindo Molecular Technologies, Inc. (Rockville,MD) Anti-PANK2, anti-MTND1, anti-NDUFA9, anti-NFS1, anti-ISCU, anti-LYRM4anti-NRF2, PDH hand complex I activity kit and aconitase kit were purchased from Abcam (Cambridge, UK), Anti-mitochondrial 10-formyltetrahydrofolate dehydrogenase (ALDH1L2), anti-alpha-aminoadipic semialdehyde synthase (AASS), anti-FOXN4, anti-hnRNPA/B,anti-NF-Y, anti-Tau, anti-GPX4 and anti-AASDHPPT were purchased from Thermo-Fisher (Waltham, MA). Anti-lipoic acid was acquired from Merck (Darmstadt, Germany). Anti-PLA2G6 and anti-SOD were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-actin was acquired from MyBiosource (San Diego, California, USA). OxyBlot Protein Oxidation Detection Kit was acquired from Merck (Darmstadt, Germany). A cocktail of protease inhibitors (complete cocktail) was purchased from Boehringer Mannheim (Indianapolis, IN). The Immun Star HRP substrate kit was from Bio-Rad Laboratories Inc. (Hercules, CA).

Labeled and unlabeled T cells were air-dried in a microscopy slide, followed by fixation in 4% paraformaldehyde and staining with Prussian Blue (Sigma, St. Louis, MO, USA) to image ferucarbotran deposits within the cell via optical microscopy. Fluorescence microscopy (Keyence Epi-Fluorescence microscope BZ-X710, Keyence, Japan) was performed using anti-dextran FITC antibody (Stemcell Technologies, Vancouver, Canada) co-incubated with T cells for 24 h in a chamber slide for live-cell imaging. Cells were stained with Hoechst 33342 for 5 minutes followed by live-cell imaging microscopy. To further evaluate ferucarbotran uptake, transmission electron microscopy of labeled T cells was performed. Cells were fixed for 24 h in a mixture of 4% glutaraldehyde and 1% paraformaldehyde. Then the sample was processed through a serial step dehydration process with ethanol, stained with osmium, and embedded in epoxy. Blocks were sectioned into 70 nm thin slices using a Leica Ultracut UCT ultramicrotome (Leica Microsystems, Wetzlar, Germany). Thin sectioned slices were dried in a copper grid prior to imaging in Hitachi H7600 electron microscope (Hitachi High-Technologies Corporation, Japan) and FIB - FEI Helios EDAX EDS/EBSD (Field Electron and Ion Company, Hillsboro, Oregon, USA).

Collagenase type V, exendin-4 (Ex4), manganese (II) chloride (MnCl₂·4H₂O, 99%), iron (III) acetylacetonate (Fe(acac)₃, 99.9%), methyl poly(ethylene glycol) (mPEG, M.W. = 2000), N-hydroxysuccinimide (NHS), N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC), oleic acid (90%), oleylamine (90%), osmium tetroxide (1%) and Prussian blue were from Sigma–Aldrich (St. Louis, MO, USA). Acryloyl chloride (96%) and N-Boc-ethylenediamine (98%) were from Alfa Aesar (Ward Hill, MA, USA). Benzyl ether and (3-aminopropyl) triethoxy silane (APTES, 98%) were from Fluka (Buchs, SG, Switzerland). N-Hydroxybenzotriazole (HOBt) and (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBOP) were from NovaBiochem (Beeston, NTH, UK). RPMI-1640 medium was from GIBCO BRL (Grand Island, NY, USA). Polyethylene (PE-50) tubing was from Clay Adams (Parsippany, NJ, USA). Guinea pig anti-swine insulin antibody was from Dako (Carpinteria, CA, USA). Rabbit polyclonal anti-SOX9 antibody (E-9) was from EMD Millipore Corporation (Temecura, CA, USA).

Immunohistochemistry of paraffin embedded, formalin fixed tissue sections of 4 µm thickness was performed. The sections were steamed for 30 minutes in a solution of citrate buffer for antigen retrieval. The primary antibodies were polyclonal rabbit anti-RFP (DsRed, PM005, MBL International), monoclonal rabbit anti-CD3 (ab16669, Abcam), mouse monoclonal anti-dextran FITC (60026Fl, Stemcell Technologies). As secondary antibody we used Cy3 donkey anti-rabbit IgG (711-165-152, Jackson ImmunoResearch Lab). Immunohistochemistry for CD3 and anti-RFP (DsRed) was performed in separate sections. Anti-RFP staining was needed because the tissue fixation and processing quenched the inherent DsRed signal from pmel DsRed T cells. Prussian Blue (Sigma, St. Louis, MO, USA) staining was also used to image iron oxide deposits within the brain tumor tissue. Fluorescence microscopy (Keyence Epi-Fluorescence microscope BZ-X710) was used for imaging.

For staining with Prussian blue (Sigma-Aldrich, Germany), promastigote and intracellular amastigotes were treated with 100 µg/mL of SPIONs for 24 h. The promastigotes (control and treated cells) were washed in phosphate-buffered saline (PBS) pH 7.2 and adhered for 10 min on glass coverslips previously coated with poly-ʟ-lysine (Sigma-Aldrich, Germany). The intracellular amastigotes were obtained after infection of RAW 264.7 macrophages at a ratio of ten parasites to one macrophage. After treatment, cells were washed in PBS pH 7.2, fixed, and dehydrated, as described in [9 (link)]. Finally, cells were observed using a DM2500 optical microscope (Leica Microsystem, Germany) in bright-field mode.