Uranyl acetate and lead citrate

To detect characteristics of differentiated odontoblast-like cells on organoids, a scanning electron microscope (SEM) and a transmission electron microscope (TEM) were used. Briefly, cultured organoids were washed with D-PBS and fixed with 2% glutaraldehyde-paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) for 12 h. After washing with 0.1 M PB, samples were post-fixed with 1% OsO4 dissolved in 0.1 M PB for 2 h, dehydrated in an ascending gradual series (50%–100%) of ethanol, infiltrated with propylene oxide, and embedded by Poly/Bed 812 kit (Polysciences, Washington, PA, USA). After pure fresh resin embedding and polymerization at 65 °C in an electron microscope oven (DOSAKA) for 24 h, they were initially cut into about 200–50 nm-thick sections and stained with toluidine blue (Sigma) for examination under a light microscope. Some 70-nm thin sections were double-stained with 6% uranyl acetate and lead citrate (Thermo Fisher Scientific, Waltham, MA, USA) for contrast staining. These sections were cut with a Leica EM UC-7 (Leica Microsystems, Tokyo, Japan) equipped with a diamond knife (Diatome, Hatfield, PA, USA) and transferred onto copper and nickel grids. All the thin sections were observed with a TEM (JEOL) at an acceleration voltage of 80 kV.