Morada ccd digital camera

Temporal lobe tissue fixed with 2.5% glutaraldehyde-2% PFA in 0.1 M phosphate buffer (PB) was transversely sectioned at 200 µm using a Leica VT1200S vibratome (Leica Microsystems). Slices were further post-fixed in 2% osmium tetroxide in 0.1 M PB for 1.5 h at room temperature, washed in deionized water and partially dehydrated in 70% ethanol. Samples were then incubated in 2% uranyl acetate in 70% ethanol in the dark for 2.5 h at 4 °C. Brain slices were further dehydrated in ethanol followed by propylene oxide and infiltrated overnight in Durcupan ACM epoxy resin (Fluka, Sigma-Aldrich). The following day, fresh resin was added and the samples were cured for 72 h at 70 °C. After resin hardening, semithin sections (1.5 µm) were obtained and lightly stained with 1% toluidine blue for light microscopy. Ultrathin sections (70–80 nm) were obtained with a diamond knife using a Ultracut UC7 ultramicrotome (Leica), stained with lead citrate and examined under a FEI Tecnai G2 Spirit transmission electron microscope at 80 kV (FEI Europe) equipped with a Morada CCD digital camera (Olympus Soft Image Solutions).

For TEM analysis, sections were embedded in epoxy resin. First, samples were post-fixed with 1% osmium tetroxide (Electron Microscopy Sciences), 7% glucose in 0.1 M PB for 30 min at room temperature, washed in deionized water, and partially dehydrated in 70% ethanol. Afterward, the samples were contrasted in 2% uranyl acetate (Electron Microscopy Sciences) in 70% ethanol for 2 h at 4°C. The samples were further dehydrated and embedded in Durcupan ACM epoxy resin at room temperature overnight, and then at 70°C for 72 h. Once the resin was polymerized, immunolabeled sections were selected and cut into semithin (1.5 μm) and then into ultrathin sections (60–80 nm) using an Ultracut UC7 ultramicrotome (Leica). Ultrathin sections were placed on formvar-coated single-slot copper grids (Electron Microscopy Sciences) stained with lead citrate and examined at 80 kV on a FEI Tecnai G² Spirit (FEI Company, Hillsboro, OR) transmission electron microscope equipped with a Morada CCD digital camera (Olympus, Tokyo, Japan).