Imaris analysis software

For immunocytochemistry (ICC), SK-N-SH cells were fixed with 4% paraformaldehyde (158127; Sigma-Aldrich, Milan, Italy), then permeabilized with Triton X-100 (T8787; Sigma-Aldrich, Milan, Italy), blocked with normal donkey serum (ab7475, abcam, Cambridge, UK), and incubated with the primary antibodies overnight at 4 °C (rabbit anti ITPKB, 12816-1-AP Proteintech, DBA, Milan, Italy, 1:1500 and mouse anti α-synuclein, Abnova, Taipei city, Taiwan, MAB5383, 1:1000). The following day, after washing with PBS, coverslips were incubated with secondary fluorescent antibodies for 2 h at RT (Alexa Fluor 488 or Alexa Fluor 555, A21206 and A31570 Thermo Fisher Scientific, Milan, Italy), washed with PBS and mounted with DAKO Fluorescence Mounting Medium (S3023; Agilent technologies, Santa Clara, CA, USA). Visualization was performed using a Leica SP8-X confocal microscope system. Using Imaris analysis software (Oxford Instruments, Abingdon, UK), the intensity of the ITPKB staining was quantified in 3D stacks of images that had been acquired with identical, non-saturating settings. After modeling the nucleus and the cytoplasm, the mean ITPKB intensity of either whole cells or nuclei/cytoplasm compartments was calculated on a 0–255 scale. Five samples were used for each case, with each sample consisting of a field with roughly 20 cells, for which intensity values were used.

Representative images for Le^X/core fucose costaining with anti-VP1 antibody (Fig. 1) were taken with the Andor Dragonfly 200 series high-speed confocal platform system (Oxford Instruments, Abingdon, UK) connected to a Leica DMi8 (Leica Microsystems, Wetzlar, Germany). Image processing and colocalization were done with the Imaris analysis software (Oxford Instruments, Abingdon, UK).
All other images were taken with a Leica DMi8 inverted fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and processed with the associated Leica Application Suite X (LAS X) software. To visualize the zebrafish, adjacent tile pictures (10% overlap) were merged together into one picture using the mosaic merge function of the LAS X software. The larvae were imaged in z-stacks and subsequently processed by the 3D-Deconvolution software of LAS X and presented as maximum projections. Quantification of fluorescent signal was done with open-source FIJI-ImageJ (79 (link)).