Luveak 812

Formalin-fixed brain samples were washed with phosphate-buffered saline (pH 7.4) and then post-fixed in 1% osmium tetroxide. Samples were dehydrated and embedded in resin Luveak-812 (Nacalai Tesque). Uranyl acetate and lead nitrate-stained ultrathin sections were then examined with a transmission electron microscope (JEM-1400Plus, JEOL, Tokyo, Japan). For negative-stain electron microscopy, samples of sarkosyl insoluble-fractions were stained with uranyl acetate, and examined with the transmission electron microscope.

Whole bladders were fixed with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer for 24 hours at 4°C. After washing in 0.1 M phosphate buffer, the samples were post-fixed with 1% OsO₄ in 0.1 M phosphate buffer for 2 hours. After dehydration with graded concentrations of ethanol and propylene oxide, the samples were penetrated and polymerized with Luveak 812 (Nacalai Tesque, Kyoto, Japan). Ultrathin 80 nm sections were cut with a microtome EM UC6 (Leica, Hiederberg, Germany), placed on mesh copper grids, stained with uranyl acetate and lead citrate, and examined at 80 kV acceleration voltage using an H7650 electron microscope (Hitachi, Tokyo, Japan).

Neutrophils (2.5 × 10⁶) were collected by centrifugation, fixed in 0.1 M phosphate buffer containing 4% paraformaldehyde/2% glutaraldehyde at 4°C, washed in isotonic phosphate-buffered sucrose, and then refixed in phosphate-buffered 1% OsO₄. Following dehydration in a graded series of ethanol washes, the cells were embedded in Luveak-812 (Nacalai Tesque). Thin sections (70–90 nm thick) were cut on an EM UC6 ultramicrotome by use of a diamond knife (Leica, Heidelberg, Germany), stained with uranyl acetate and lead citrate and then observed under an H-7650 electron microscope (Hitachi).

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.

Acute myeloid leukemia cells were collected by centrifugation, fixed in 0.1 m phosphate buffer containing 4% paraformaldehyde/2% glutaraldehyde at 4 °C, washed in isotonic phosphate‐buffered sucrose, and then refixed in phosphate‐buffered 1% OsO₄. Following dehydration in a graded series of EtOH washes, the cells were embedded in LUVEAK‐812 (Nacalai Tesque). Thin sections (70–90 nm thick) were cut on EM UC6 Ultramicrotome using a diamond knife (Leica, Heidelberg, Germany), stained with uranyl acetate and lead citrate, and then observed under H‐7650 electron microscope (Hitachi, Tokyo, Japan).

After washing with PBS, day 13 + 28 kidney organoids and mouse embryonic kidneys were fixed with 1.4% PFA/1% glutaraldehyde/0.1 M PB overnight at 4 °C. Then, the organoids were washed with isotonic phosphate-buffered sucrose, refixed with phosphate-buffered 1% OsO₄, dehydrated through a graded series of ethanol solutions, and embedded in Luveak 812 (Nacalai Tesque). Thin sections (70–90 nm thick) were cut with a diamond knife on an EM UC7 ultramicrotome (Leica), stained with uranyl acetate and lead citrate, and observed using a JEM-1400Flash electron microscope (JEOL).