Immunofluorescence Imaging Protocol

For immunofluorescence, cells were fixed in 4% paraformaldehyde and processed as previously described (Vagnarelli et al., 2011 (link)). Fluorescently labelled secondary antibodies were applied at 1:200 (Jackson ImmunoResearch, 715-585-150 and 715-096-150 ). Three-dimensional data sets were acquired using a cooled CCD camera (CH350, Photometrics) on a wide-field microscope (DeltaVision Spectris; Applied Precision) with a NA 1.4 Plan Apochromat lens. The data sets were deconvolved with softWoRx software (Applied Precision). The three-dimensional data sets were then converted to Quick Projections in softWoRx. Three-dimensional data sets were also acquired using a wide-field microscope (Nikon Ti-E super research Live Cell imaging system) with a NA 1.45 Plan Apochromat lens. The data sets were deconvolved with NIS Elements AR analysis software (Nikon). The three-dimensional data sets were converted to Maximum Projections using the NIS software. In both cases, the images were exported as TIFF files, imported into Adobe Photoshop and then imported into Inkscape for final presentation.