Tecnai g2 bio twin electron microscope

The 96 hpf larvae were fixed with 2.5% (w/v) glutaraldehyde in 0.1 M, pH 7.4 sodium cacodylate buffer for two days at 4°C. The samples were washed three times 5 min in the same buffer. Samples were treated to 1% (w/v) osmium tetroxyde in cacodylate buffer for 1h, then dehydrated through a graded ethanol series, and finally embedded in monomeric resin (Epon 812). All chemicals used for histological preparation were purchased from Electron Microscopy Sciences (Hatfield, USA). Sections for optical and electron microscope of 500 and 80 nm respectively were obtained with an ultramicrotome UCT (Leica Microsystems GmbH, Wetzlar, Germany). The semi-thin sections were stained with aqueous blue toluidine and examined under a light microscope (Leica, DM750) equipped with a Leica camera ICC50 and the LAS EZ Software. For TEM analysis, ultrathin sections were mounted on copper grids and examined in a Tecnai G² Biotwin Electron Microscope (FEI Company, Eindhoven, the Netherlands) using an accelerating voltage of 100 kV and equiped with a CCD camera Megaview III (Olympus Soft imaging Solutions GmbH, Münster, Germany). For each replicate, at least 20 micrographs of local detailed structures were taken, analysed and compared for each condition.