1400 transmission electron microscope

The specimens were fixed in cold 2.5% glutaraldehyde in PBS, pH 7.3. The specimens were rinsed in PBS, post-fixed in 1% osmium tetroxide with 1% potassium ferricyanide, dehydrated through a graded series of ethanol (30–90%) and embedded in Poly/Bed® 812 (Luft formulations). Semi-thin (300 nm) sections were cut on a Leica Reichart Ultracut (Leica Microsystems, Buffalo Grove, IL), stained with 0.5% Toluidine Blue in 1% sodium borate and examined under the light microscope. Ultrathin Sects. (65 nm) were stained with 2% uranyl acetate and Reynold’s lead citrate and examined on JEOL 1400 transmission electron microscope (JEOL Peabody, MA) (NIH grant #1S10RR016236-01, Simon Watkins) with a side mount AMT 2 k digital camera (Advanced Microscopy Techniques, Danvers, MA).

At the end of each study, two tissue wedges containing the TM and SC were dissected 180° apart from each anterior segment and fixed in 10% neutral buffered formalin. Tissue wedges were post fixed in 2% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA) in 0.1 M phosphate buffer followed by dehydration in ascending ethanol concentrations. Tissues were subjected to a clearing agent (acetone, Sigma-Aldrich), embedded in epoxy resin blocks, and 500 nm and 100 nm sections were obtained using an ultramicrotome (Leica Microsystems, Buffalo Grove, IL). 500 nm sections were stained with toluidine blue and gross morphology was assessed by light microscopy. 100 nm sections were placed on copper grids and stained with 2% uranyl acetate (Electron Microscopy Sciences) followed by lead citrate (Mager Scientific, Dexter, MI). Sections were imaged using a JEOL 1400 transmission electron microscope (JEOL USA, Peabody, MA) for evaluation of cell and tissue ultrastructure.

EM reconstruction was done in three to five mice from each genotype (NTG1, APP, PS1, APP/PS1, NTG2, 3xTgAD, Tau). For TEM, mice were anesthetized with either intraperitoneal injection of ketamine/xylazine or inhalation of a mixture of 5% isoflurane and oxygen followed by cardio perfusion with 4% paraformaldehyde (PFA). Whole brains were removed and post-fixed in Trump’s solution (4% formaldehyde + 0.1% glutaraldehyde in 0.1M phosphate buffer) overnight at room temperature. The hippocampus (CA1 region) was dissected from each hemisphere and processed for TEM. Tissue was fixed in 1% osmium tetroxide and 1% aqueous uranyl acetate, dehydrated in a graded series of ethanol, and embedded in Embed 812/Araldite (EMS, Hatfield, PA). Thin sections (0.1 μm) were collected on copper grids, post-stained with lead citrate and viewed at 80 kV with a JEOL 1400 transmission electron microscope (JEOL USA, Peabody, MA). Ten random areas from each CA1 region were imaged, and only neuropils longer than 3 μm were selected for analyses. Mitochondrial profiles were scored according to their appearance as regular elongated (>3 μm long), ovoid (0.5 μm in diameter), teardrop shaped, or MOAS. Analyses were done by investigators blinded to the mouse genotype.

Exosomes were prepared as described by Walther and Ziegler (2002) with minor modifications. Samples were high pressure frozen, freeze substituted and embedded in Epon. Ultrathin sections were cut with an ultramicrotome and visualised with a Jeol 1400 transmission electron microscope (Jeol Inc.) [67 (link)].