Ruby ccd camera

Astrocytes were grown on translucent Corning polyethylene terephthalate Transwell permeable supports (Merck, Darmstadt, Germany). Controls and Rapa-treated cells were fixed using modified Karnovsky fixative. After post-fixation with 1% OsO₄ and dehydration in a series of ethanol solutions, astrocytes were embedded in EPON resin (Serva, Heidelberg, Germany). Ultrathin sections were contrasted with 3% lead citrate and 0.5% uranyl acetate using an automatic contrasting system (EM AC 20; Leica, Wetzlar, Germany). Sample imaging was performed at 120 kV using TEM JEM-1400 Plus (JEOL, Tokyo, Japan) and Ruby CCD camera (JEOL, Tokyo, Japan).

Eggs were squeezed from gravid females as described previously (Westerfield, 2000 ), stored in Hank's medium and fixed overnight at 4°C in 2.5% (v/v) glutaraldehyde with 0.1 M PIPES buffer (pH 7.2) complemented with Hank's salts. After squeezing, females (two wild types and one double mutant) were killed using excess anaesthetic, whole ovaries were dissected as described (Elkouby and Mullins, 2017 (link)) and fixed as above. The next day, samples were washed briefly with 0.1 M PIPES buffer (pH 7.2) and post-fixed in 1% (v/v) osmium tetroxide in water. Samples were then dehydrated through a graded ethanol series before infiltration with Spurr resin (TAAB) and polymerization at 60°C for 24 h. Semi-thin sections were cut at ∼450 nm and stained with 1% Toluidine Blue. Ultrathin sections (80 nm) were cut using an ultramicrotome (UC 7, Leica Microsystems), mounted on grids and post-stained with Uranyless stain (TAAB S474) and Reynolds lead citrate (Reynolds, 1963 (link)). Samples were examined using a TEM operated at 120 Kv (JEOL JEM 1400Plus, JEOL, Japan). Images were acquired with a 2k by 2k format CCD camera (JEOL Ruby CCD Camera). At least ten squeezed eggs and five oocytes at each developmental stage (identified according to Selman et al., 1993 (link)) were examined in each sample and the phenotype in wild-type or mutant samples matched the results shown in all cases.

Five microliters of the protein solution were applied to glow-discharged copper grids with continuous carbon film (20 nm thickness, prepared in the lab) after heat shock. Excess solution was blotted from the grid after 60 s adsorption time, and 5 μl stain solution (2% wt uranyl acetate) were applied immediately. After 30 s, the remaining stain solution was blotted away and the grid was allowed to dry. The grids were transferred to a JEOL JEM-1400Plus microscope equipped with a JEOL Ruby CCD camera and image acquisition was performed at 120 kV acceleration voltage at nominal magnifications of 10k and 60k.

To study the interaction between AuNP and neurons, dissociated hippocampal neurons (7 DIV) grown on polylysine-coated Thermanox coverslips (Thermo Fisher UK), were treated with AuNP for 24 h, fixed, osmicated, dried, embedded in Taab 812 resin (Sigma-Aldrich, UK) and sectioned to 70 nm as described in our previous article.^{35 (link)} Sections were immediately collected on 2 × 1 mm slot grids formvar and carbon coated TEM copper grids (Agar Scientific, UK), dried and stored until TEM analysis. TEM imaging of hippocampal neurons was done at 120 kV (JEOL JEM 1400Plus, JEOL, Japan) with a 2k × 2k format CCD camera (Ruby CCD Camera, JEOL, Japan) as previously described.^{35 (link)}

For electron microscopy, cells were first grown in synthetic complete medium containing glucose as the carbon source and then shifted to synthetic complete medium containing galactose as carbon source, in which they were grown overnight. Chemical fixation of yeast cells with glutaraldehyde and osmium tetroxide, dehydration, Epon embedding and subsequent steps of sample preparation for electron microscopy were performed as described previously (Unger et al., 2017 (link)). Electron micrographs were taken with a JEOL JEM-1400 Plus transmission electron microscope operated at 80 kV, a JEOL Ruby CCD camera (3296×2472 pixels), and the TEM Center software Ver.1.7.12.1984 or Ver.1.7.19.2439 (JEOL, Tokyo, Japan).