Technai g2 spirit biotwin transmission electron microscope

Poliovirus stocks were purified by cesium chloride gradient centrifugation and were concentrated and desalted using Amicon filters (Millipore) as previously described (43 (link)). Poliovirus samples containing 9.3 × 10⁶ PFU were inactivated by treating with 2.5% glutaraldehyde for 1 h at room temperature. After inactivation, 2.5 µl of the inactivated virus was placed on 400-mesh carbon-coated copper grids that had been glow discharged for 30 s using PELCO EasiGlow 91000. The grids were stained with 2% phosphotungstic acid and examined using a TEI Technai G² Spirit Biotwin transmission electron microscope (FEI, Hillsboro, OR) equipped with a Gatan ultrascan charge-coupled-device (CCD) camera, operated at an acceleration voltage of 120 kV. Images were taken at magnifications of ×13,000 and ×30,000.

The scutellum of 2 pharate adults, 4 just emerged adults (2 females and 2 males), 4 three day old adults (2 females and 2 males) and 8 ten-day-old adults (4 females and 4 males) of B. oleae were dissected from anaesthetized insects and fixed for 3 h in 2.5% glutaraldehyde in cacodylate buffer (Electron Microscopy Sciences, Hatfield, England), pH 7.2. The fixed scutelli were repeatedly rinsed in sodium cacodylate buffer and post-fixed for 1 h at 4 °C in 1% osmium tetroxide in sodium cacodylate buffer (Electron Microscopy Sciences). The samples were then repeatedly washed in the same buffer, dehydrated in ascending ethanol concentrations and finally embedded in an Epon-Araldite resin mixture (Sigma-Aldrich). Afterwards, ultra-thin sections were cut using a Leica EM UC6 ultramicrotome (Leica Microsystem GmbH, Wetzlar, Germany), collected on Parlodion (Spi-Chem, West Chester, PA, USA) coated copper grids, stained with uranyl acetate and lead citrate (Electron Microscopy Sciences, Hatfield, England) and examined using a Technai G2 Spirit BioTwin Transmission Electron Microscope (FEI company, Hillsboro, OR, USA) equipped with a CCD camera Veleta (EMSIS) 2048 × 2040.