Tecnai g2 12 microscope

AgNPs morphology, size and stability over time in the culture medium, were analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). TEM analysis was performed with a Fei Tecnai 12 G2 microscope operated at 120 kV. NPs were suspended in ultrapure water at 100 mg/l, deposited on carbon coated copper grids and left to dry overnight. Micrographs were recorded on a side mounted Olympus Morada CCD camera. Size distribution analysis was performed using a semi-automatic method implemented in ImageJ software (Schneider et al., 2012 (link)). The analysis involved particle segmentation and area determination. The diameter of each particle was estimated from the particle area assuming a perfectly circular particle. For DLS analyses, AgNPs 50 mg/l suspension in ultrapure water was analyzed with a Malvern Zetasizer Nano ZS DLS instrument operated in backscattering mode. Measurements were carried out at 23°C and were conducted in triplicate to check for sedimentation and solution stability.

Eye discs WT or mutant for Stam, Hrs, Stam l(2)gl or Vps25 were fixed in 2.5% glutaraldeyde diluted in 0.1 M sodium cacodylate buffer for 3 hours at room temperature. Eye discs were post-fixed in 1% osmium tetroxide (Electron Microscopy Science, Hatfield, PA, USA) for 2 hours at room temperature and subsequently in 1% uranyl acetate (Electron microscopy science) for 1 hour. Samples were dehydrated through a graded ethanol series and next in propylene oxide before embedding in epoxy resin (Poly-Bed, Polyscience, Warrington, PA, USA) overnight at 42°C and then 2 days at 60°C. Searching for the eye disc epithelium was performed on semi-thin sections (500 nm) stained with toluidine blue. Ultrathin sections of 50 nm were then cut and stained with 5% uranyl acetate and lead citrate. Representative TEM micrographs of each sample were taken with Tecnai 12-G2 microscope (FEI company, Eindhoven, The Netherlands) and processed with Adobe Illustrator CS5. Quantifications were performed with Image J on an set of approximately 20 micrographs per sample.