Immunofluorescence Imaging of HCMV Infection

Indirect immunofluorescence analysis of HFF cells was performed by fixation with 4% paraformaldehyde and fluorescence staining as described elsewhere (Scherer et al., 2014 (link)). When late stages of HCMV infection were investigated, an additional blocking step was performed by incubating cells with 2 mg/ml γ-globulins from human blood (Sigma) for 30 min at 37 °C, before primary antibodies were applied. Confocal images were obtained with a Leica TCS SP5 confocal microscope or a Zeiss Axio Observer Z1 equipped with an Apotome.2. The images were processed with Adobe Photoshop CS5 or ZEN 2.3 and assembled using CorelDraw. For 3D reconstruction of z-series images, Huygens Professional Software (Scientific Volume Imaging) was used. Stacks of confocal images from single cells were imaged, deconvoluted using the Huygens Deconvolution wizard and 3D images generated with the Huygens Surface Renderer tool. For quantifications, z-series images (0.3–0.7 µm distance) of at least 50 cell nuclei per sample were taken. To measure number and size (perimeter) of PML foci, automated ImageJ-based quantification was performed on maximum intensity projection images 3D animations were generated from z-series images using the Leica LAS AF software.