Jfc 1300 sputter coater

At 18 hpi, control and ECTV-infected L929 and RAW 264.7 cells grown on glass coverslips in a 24-well plate were fixed for 1 h with 2.5% glutaraldehyde in phosphate buffer. Next, cells were rinsed in phosphate buffer and post-fixed for 1 h with 1% osmium tetroxide in phosphate buffer. Fixed cells were dehydrated for 10 min at RT in each solution of ethanol with increasing concentrations (70% and 95%), twice in absolute ethanol for 10 min at RT, and twice in acetone for 10 min at RT. Next, samples were dried using a CPD 7501 critical point drier (Polaron, Hatfield, PA, USA) and coated with a gold layer in a JFC-1300 sputter-coater (JEOL Ltd.). For analysis of specimens, the FEI Quanta 200 environmental scanning electron microscope (ESEM) with EDAX energy dispersive spectroscopy (EDS) system (FEI, Tokyo, Japan) was used.

GM-BM were seeded on microscopic slides placed in a 24-well plate at a density of 2 x 10⁵ cells/well. Cells were left uninfected or were treated with live-ECTV or uvi-ECTV for 60 min at 37°C, and then incubated in the absence or the presence of LPS for 24h. Next, GM-BM grown on microscopic slides were fixed for 60 min with 2.5% glutaraldehyde in phosphate buffer and postfixed for 60 min with 1% osmium tetroxide in phosphate buffer. After dehydration in ethanol and acetone series and drying in a CPD 7501 critical point drier (Polaron; Hatfield, PA, USA), GM-BM were coated with a gold layer in a JFC-1300 sputter-coater (JEOL, Tokyo, Japan). The SEM imaging was operated under FEI Quanta 200 environmental scanning electron microscope (ESEM) with EDAX EDS system (FEI, Tokyo, Japan).