Ipgell pg20 1

For immunoelectron microscopy, β2m KO splenic DCs (3 × 10⁶) were cultured with UV (500 mJ/cm²)-irradiated apoptotic K^b mouse splenocytes (3 × 10⁷) in a microtube for 1 h at 37°C. Then DCs were re-purified using anti-CD11c microbeads (130-125-835, Miltenyi Biotech), and stained with biotinylated anti-K^b mAb (clone AF6-88.5, BioLegend), followed by streptavidin-10 nm gold particles (S9059, Merk). Cells were then embedded in iPGell (PG20-1, GenoStaff) according to the manufacturer’s instructions. The cell blocks were fixed with 4% formaldehyde and 2% glutaraldehyde in phosphate-buffered saline (PBS; pH7.4) overnight at 4°C, then were postfixed with 1% OsO₄ in PBS for 2 h. Following dehydration in a series of graded concentrations of ethanol, the fixed cell blocks were embedded in epoxy resin (Luveak 812; Nacalai Tesque). Ultrathin sections (70-nm thickness) were prepared on an ultramicrotome (EM UC7; Leica). The sections were then stained with uranyl acetate and lead citrate and finally examined with an electron microscope (JEM-1400Flash, JEOL Ltd.). For the standard electron microscopy, live, UV (500 mJ/cm²)-irradiated, f-t splenocytes and SCT/BW5147 cells were fixed and analyzed as described above. TEM was performed at the Division of Electron Microscopic Study, Center for Anatomical Studies, Graduate School of Medicine, Kyoto University.

The hiPSC-NS/PCs cells were cultured for 5 days in a floating culture, and the neurospheres were administered rhHGF (20 ng/per) or PBS. After fifteen minutes, it embedded in iPGell (PG20-1; Genostaff, Tokyo, Japan) following the manufacturer’s instructions, and frozen and sectioned at a 16 μm thickness on a cryostat (CM3050S; Leica Microsystems, Wetzlar, Germany). Tissue sections were stained with the following primary antibodies: anti-SOX2 (mouse IgG2a, 1: 40, RD systems, Inc., MAB2018), anti-Nestin (mouse IgG1, 1: 200, Millipore, Inc., MA5326), anti-c-Met (mouseIgG1, 1: 200, Cell signaling, Inc., 25H2), anti-phosphorylated c-Met (rabbit IgG, 1:100, Invitrogen, Inc., 44-882G). All samples were incubated overnight at 4 ℃ and then incubated with Alexa Fluor-conjugated secondary antibodies (1: 500, Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Nuclei were stained with Hoechst 33,258 (10 μg/ml, Sigma-Aldrich), then examined under a confocal laser-scanning microscope (LSM 700, Carl Zeiss, Jena, Germany).