Mouse monoclonal anti fth1

After fixation and dehydration, the samples were incubated with propylene oxide prior to exposure to propylene oxide/Epikote [13 (link),14 (link)]. The blocks were embedded with Epon-Araldite mixture (Electron Microscopy Sciences, Hatfield, PA, USA) and sectioned. The sliced samples were then examined under a transmission electron microscope (HITACHI-7000, Hitachi, Japan). For immunogold labeling, the sections of the cells on nickel grids were treated with 10% H₂O₂ for 10 min and then blocked with SuperBlock^TM blocking buffer (Thermo Fisher Scientific Inc.) for 30 min followed by mouse monoclonal anti-FTH1 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and rabbit polyclonal anti-NCOA4 (Abcam) antibody treatment overnight. The grids were hybridized with goat polyclonal anti-mouse (20 nm) and goat polyclonal anti-rabbit (12 nm) secondary antibodies containing gold particles (Abcam) for 1 h. The sections were then stained with saturated uranyl acetate and lead citrate. The images were examined under a transmission electron microscope.

After treatment, cells fixed with paraformaldehyde were permeabilized with 0.1% Triton X-100 [15 (link)]. After washing with TBST and blocking with 5% BSA, the cells were stained with mouse monoclonal anti-FTH1 (Santa Cruz Biotechnology, Inc.) or anti-NCOA4 antibody in combination with rabbit polyclonal anti-LC3 (Abcam) antibody. After washing, the cells were stained with Alexa Fluor^® 568 goat anti-mouse IgG and Alexa Fluor^® 488 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA). Nuclei were visualized by incubating the cells with DAPI. The signal was examined using a confocal microscope (Zeiss LSM780, Zeiss, Oberkochen, Germany). For autophagic flux study, cells were transfected with tandem fluorescent-tagged LC3 (mRFP-GFP) reporter plasmid (ptfLC3) [16 (link)]. The signal was examined using fluorescence microscopy (Olympus BX61, Tokyo, Japan).