Cm100

U2OS cells were transfected with control siRNA or ATG13 siRNA for 48 h and infected with CV for 7 h at an MOI of 1. After a 2-h fixation at room temperature in 2% PFA and 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer, pH 7.4, cells were embedded in epon resin as previously described (Verheije et al., 2008 (link)). Subsequently, 70-nm sections were obtained using an UC7 Leica Ultra-microtome and stained with uranyl acetate and lead citrate as previously described (Verheije et al., 2008 (link)). Cell sections were analyzed using an 80-kV transmission electron microscope (CM100; FEI). Two independent grids were used to perform the counting analysis of 125 cells per condition in total. Three different categories of membranous rearrangements were identified in CV-infected cells and defined as follows: category 1, closely packed, elongated, regular shaped single-, double-, or multimembrane vesicles; category 2, closely packed, irregular shaped, single-membrane vesicles with a light content; and category 3, irregular, collapsed membrane clusters.

Vascular graft samples were fixed in 3.0% glutaraldehyde overnight, post-fixated in Palade’s osmium tetroxide, and dehydrated in a graded acetone series [15 (link)]. Dehydrated samples were impregnated/embedded in epoxy to facilitate the making of ultra-thin sections for the TEM evaluation. Ultra-thin sections were cut from the sample embedded in the epoxy using an ultra-microtome (Leica Ultracut UC7, Vienna, Austria). After sectioning the samples, they were stained with uranyl acetate and lead citrate. Sections of the leaflet samples were evaluated by using a transmission electron microscope (CM100, FEI, The Netherlands) and photographed using an Olympus Soft Imaging System Megaview III digital camera with Soft Imaging System digital image analysis and documentation software (Olympus, Tokyo, Japan).