Alkaline lead citrate

The OC-1 cells were fixed in 2.5% glutaraldehyde (Sigma-Aldrich) for 24 h and 1% osmic acid (Sigma-Aldrich) for 1–2 h, dehydrated with acetone (Sinopharm Chemical Reagent), and embedded with Araldite CY212 (TAAB). Ultrathin sections were stained with alcohol uranyl acetate (Polysciences) and alkaline lead citrate (Sigma-Aldrich). The sections were gently washed with distilled water and observed under a JEM 1230 transmission electron microscope (JEOL Ltd.).

The mouse brains were fixed in 2.5% glutaraldehyde (P1126, Solarbio, BeiJing, China) for 24 h, immobilized in 1% osmic acid (SigmaAldrich, O5500) for 1–2 h, dehydrated with acetone (Sinopharm Chemical Reagent, u1006801), and embedded in araldite CY 212(TAAB, E009). Ultrathin sections were stained with alcoholic uranyl acetate (Polysciences, 6159-44-0) and alkaline lead citrate (SigmaAldrich, 15326). The sections were washed gently with distilled water and observed with a JEM 1230 TEM (JEOL Ltd., Tokyo, Japan).

For electron microscopic analyses, different cell lines were fixed in freshly prepared 2% glutaraldehyde ELMI grade in phosphate buffered saline (PBS; both Sigma-Aldrich) for 2 hours at 4 °C, washed and stored in PBS. Subsequently, samples were post-fixed in 1% veronal-acetate-buffered OsO₄ (Merck) for 1 hour, dehydrated in a graded series of ethanol and embedded in epoxy resin (Serva). Ultrathin sections were stained in 1% uranyl acetate or alternatively, in 0.2% OTE (Oolong Tea Extract, Nisshin EM Co.), and subsequently in 8% alkaline lead citrate (both Merck), followed by examination in a transmission electron microscope at 80 kV (Tecnai 20, FEI Company).