Reynolds lead citrate

Cells were postfixed with 1% osmium tetroxide (OsO₄) and 1% potassium ferrocyanide for 2 h at 4 °C. Cells were dehydrated with increasing concentrations of ethanol ending in acetone and embedded in Agar 100 resin (Agar Scientific Ltd., UK). Resin-embedded cell cultures were cut perpendicular to the plane of the Thermanox™ surface using a Leica UC60 ultramicrotome. The obtained ultrathin sections (~ 60 nm) were collected on Formvar-coated grids and counterstained with 1% uranyl acetate and Reynolds’ lead citrate (Agar Scientific Ltd., UK) and then examined using a Zeiss Leo 912 AB transmission electron microscope (Zeiss, Germany) operated at 120 kV. Digital images were recorded with a Veleta CCD camera. The ESIvision software was used for image capture and processing.

The 1 mm³ fragments of brain tissue were fixed by immersion in 4% glutaraldehyde, buffered with 0.1 mol L⁻¹ sodium cacodylate (pH 7.3) at 4°C overnight and 25 pieces were further processed for Epon‐embedding (Agar100 resin, Agar Scientific, Essex, UK) as previously described.15 Epon‐embedded murine cerebral tissue fragments were sectioned using a Leica EM UC7 ultramicrotome (Leica Microsystems GmbH, Vienna, Austria). Light microscopy was done on 1‐μm‐thick sections stained with 1% toluidine blue and representative images were recorded under a Nikon 600 light microscope performed with a 100× objective (oil immersion) with a 11‐megapixel camera (AxioCam HRc, Zeiss, Germany). Random areas from the brain tissue of both 6‐month and 24‐month‐old mice were oriented for ultrastructural analysis. Five Epon‐embedded blocks from each mouse were sectioned for TEM and mounted on 50‐mesh copper grids (Agar Scientific). Electron microscopy imaging was performed on 60 nm ultra‐thin sections counterstained with uranyl acetate and Reynolds lead citrate (Agar Scientific) at 80 kV on a Morgagni 268 TEM (FEI Company, Eindhoven, The Netherlands), equipped with a MegaView III CCD (Olympus, Germany) and running iTEM‐SIS software (Soft Imaging Systems, Olympus, Germany).

For room-temperature transmission electron microscopy (TEM), cells were fixed for 2 h with a pre-warmed solution comprising 2.5% glutaraldehyde and 1.4% sucrose (pH 7.2); then, they were processed for epoxy resin embedding, as detailed by the manufacturer (Agar100 Resin Kit, Agar Scientific, Essex, UK) and as described in previous work from this group [21 (link)]. Sectioning was performed on a Leica EM UC7 ultramicrotome (Leica, Wetzlar, Germany), and the ultra-thin sections (40–60 nm) were mounted on carbon and Formvar-coated 50-mesh copper grids and counterstained with 1% uranyl acetate and Reynolds lead citrate (Agar Scientific, Essex, UK). Imaging was done using a 200 kV Talos F200C TEM, equipped with a 4K × 4K Ceta camera (Thermo Fisher Scientific, Waltham, MA, USA).