Jsm 1400 electron microscope

The fixation protocol followed an established procedure (Schikorski & Stevens, 1997). Briefly, anesthetized WT and Rab27B knockout mice were perfused with oxygenated saline followed by 10 min perfusion with 4% glutaraldehyde in 100 mM phosphate buffer, pH 7.2. The brain was removed and fixed overnight in the same fixative. After rinsing, 300 µm‐thick Vibratome slices of the brain, cut through the hippocampus region, were post‐fixed for 1 hr at 4°C in a mixture of 1% OsO₄ and 1.5% K⁺‐ferrocyanide. The slices were rinsed, bisected to make half brains, dehydrated and flat embedded in Epon. Ultrathin sections of the CA1 region where examined after staining with lead citrate and uranyl acetate with a JEOL JSM 1400 electron microscope. Images of synapses were recorded digitally at a print magnification of 68,900× and processed in Adobe Photoshop CS6 (Adobe Photoshop, RRID:SCR_014199). Synaptic vesicle diameters were determined using the Photoshop measurement software. Synaptic vesicles were analyzed in synapses of 2 WT and 2 Rab27B knockout mice. For CA1 a total of 17 WT and 18 KO synapses were analyzed and diameters of 113 WT and 130 Rab27B KO synaptic vesicles were measured. In CA3, 221 WT and 234 Rab27 KO synaptic vesicles were measured from 22 and 17 synapses, respectively.

Triplicates of control and TM-treated cells cultured in monolayers in a six-well plate, were fixed with 4% PFA, 2.5% glutaraldehyde, and 0.002% picric acid in 0.1 M sodium cacodylate buffer, pH 7.3 overnight at 4 °C. Samples were washed with buffer and post-fixed in aqueous 1% OsO₄, 1.5% potassium ferricyanide for 1 h, washed with buffer. After a wash in deionized water, the samples were en bloc stained with 1.5% uranyl acetate(aqueous) for 1 h in the dark. Samples were dehydrated through a graded ethanol series then infiltrated with epoxy resin (LX112, Ladd Research Industries). Polymerized blocks were trimmed and sectioned at 65 nm. Sections were mounted on 200 mesh, thin-bar copper grids and post-stained with lead citrate. Samples were viewed on a JEOL JSM 1400 electron microscope (JEOL, US, Peabody, MA) and images were captured on a Veleta 2K × 2K CCD camera (EMSIS, GmbH, Muenster, Germany). Quantification of abnormal cristae/mitochondrial area was done using ImageJ software.