Epon812

Mouse and human brain samples were fixed with 2.5% glutaraldehyde in 0.1 M phosphate-buffered saline (PBS) for 2 h, incubated with 1% OsO4 buffered with 0.1 M PBS for 2 h, and dehydrated in a series of graded concentrations of ethanol (50, 70, 80, 90, 100, 100, 100, and 100%), and embedded in Epon812 (E14120, science services, München, Germany). Semi-thin (1 μm) sections for light microscopy were collected on glass slides and stained for 30 s with toluidine blue. Ultrathin (90 nm) sections were collected on copper grids, double-stained with uranyl acetate and lead citrate. Images were obtained by transmission electron microscopy (H-7100, Hitachi, Hitachinaka, Ibaraki, Japan).
Human samples (cerebral neocortex) was collected at autopsy and directly fixed in 4% paraformaldehyde two overnight. The sliced brain samples were preserved in 20% sucrose contained phosphate buffer in 4 °C until the process for ultrastructural observation. Following procedures were same as mice tissue sample preparation.

Light microscopy images were obtained with an Olympus Epi-Fluorescence Microscope (BX-51) essentially as described previously (Lu et al., 2017) (link). Samples for TEM were prepared using published procedures (Lu et al., 2017 (link); Armarego-Marriott et al., 2019) (link) with minor modifications. Briefly, leaf samples were fixed in 2.5% (v/v) glutaraldehyde in 50 mM sodium cacodylate (pH 7.4) containing 5 mM CaCl₂ for 1 h under vacuum. Fixation was continued at 4°C overnight, and followed by post-fixation with 1% (w/v) OsO₄ and 0.8% (w/v) K₃Fe(CN)₆ in 50 mM cacodylate buffer (pH 7.4) for 2 h at 4°C. After rinsing the leaf samples, en bloc staining of the tissue was performed by incubation in 2% (w/v) aqueous uranyl acetate for 2 h at root temperature. Following dehydration in acetone, embedding in Epon-812 (Science Services GmbH, Munich, Germany) was carried out using standard protocols. For electron microscopy, ultrathin sections (50–70 nm) were cut with diamond knives, contrasted with 2% (w/v) uranyl acetate and lead citrate, and examined in a Zeiss EM 912 Omega transmission electron microscope (Carl Zeiss, Oberkochen, Germany).