Jem 1400 plus 120 kv transmission electron microscope

Samples containing L1 worms in fed, starved, CeMM, and refed OP50 conditions were prepared and subjected to transmission electron microscopy analyses as described [71 (link)]. After three washes in M9, worms were resuspended in M9 containing 25 mM levamisole and 20% BSA. Samples were processed to cut thin sections onto standard 200 mesh grids and poststained with uranyl acetate and lead citrate. Images were taken with a JEOL JEM-1400Plus 120kV Transmission Electron Microscope (JEOL, Tokyo, Japan) equipped with an AMT XR111 high-speed 4,000 × 2,000 pixel phosphor-scintillated 12-bit CCD camera.

Electron microscopy was performed at the Indiana University Electron Microscopy Center. Samples were fixed in 2.0% glutaraldehyde–1% tannic acid in unsupplemented PM medium for 1 h at room temperature, with the exception of Rvu. sulfidophilum, which was fixed in 2.0% glutaraldehyde in modRM2 medium containing 2% (wt/vol) NaCl. Fixed samples were placed on ice and put through four changes of unsupplemented medium, with incubation in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.2) for 1 h, followed by two changes of sodium cacodylate buffer. Samples were then put through a graded ethanol dehydration series to 100% ethanol with en bloc staining of 2% uranyl acetate for 30 min at the 75% ethanol step. Samples were subsequently put through three changes of 100% ethanol at room temperature, followed by infiltration using low-viscosity embedding resin (Electron Microscopy Sciences, Hatfield, PA) with four changes of resin before the samples were polymerized at 65°C for 18 h. Ultrathin sections of 85-nm thickness on 300 mesh copper grids were obtained using a Leica Ultracut UCT ultramicrotome. Grids were stained with saturated uranyl acetate and lead citrate and imaged using a JEM-1400Plus 120-kV transmission electron microscope (JEOL United States, Inc.) with a Gatan OneView 4K by 4K camera.

TEM examination was performed using a JEM-1400(PLUS) 120 kV transmission electron microscope (JEOL GmbH, Japan). For the sample preparation, we used the nanoparticle suspension before freeze-drying and put a few drops of this on a carbon-coated copper grid.