Quetol 812 mixture

Fluorescein isothiocyanate-labeled Con A was added to the cells at the pairing stage (16 h after the mixing) at a final concentration of 10 μg mL^-1, followed by incubation for 4 h under continuous light. The cells were then harvested by centrifugation, prefixed in 2% glutaraldehyde in 0.1 M phosphate buffer or 0.05 M Cacodylate buffer (pH 7.2), and kept at room temperate for 2 h. Next, they were gently centrifuged, washed three times in this buffer, and postfixed in 1% OsO₄ with the same buffer at 4°C overnight. After washing with the buffer, 1 × 10⁵ cells were collected by filtration using SUPREC-01 (Takara, Shiga, Japan). They were embedded in 2% low-melting-point agarose, dehydrated through an ethanol series, and passed through QY-2 (methyl glycidyl ether; Nisshin EM, Tokyo, Japan) and embedded in Quetol-812 mixture (Nisshin EM), followed by polymerization at 60°C for 48 h. Ultrathin sections of 60–70 nm were cut with an ultramicrotome, Ultracut S (Reichert-Nissei, Tokyo, Japan), stained with 4% uranyl acetate and 0.4% lead citrate, and observed with a TEM, JEM-1200EXS (JEOL, Tokyo, Japan), at 80 kV.

Kidneys from 16-week-old mice were cut into small pieces (1 mm³) and prefixed in 2.5 % buffered glutaraldehyde for 4 h, and then fixed with 1 % buffered osmium tetroxide for 2 h. Fixed tissues were dehydrated by graded alcohol, and embedded in epoxy resin (Quetol 812 Mixture; Nisshin EM, Tokyo, Japan). Epoxy resin-embedded specimens were sectioned at a thickness of 70 nm, stained with uranyl acetate and lead citrate, and observed under a JEOL transmission electron microscope (JEM-1210; JEOL, Tokyo, Japan).