Cis golgi

Cells were seeded onto 96-well glass-bottomed plates (Cellvis) at 70% confluency. The following day cells were fixed with 4% paraformaldehyde (PFA) (Affymetrix), washed three times in PBS and then permeabilised with 0.2% Triton X-100 (Sigma). Cells were blocked in 2% w/v bovine serum albumin (BSA) in PBS for 30 min before incubation with primary antibodies for 1 h at room temperature. Plates were then washed in PBS before being incubated with secondary antibodies for 1 h at room temperature in the dark. After washing again in PBS, cells were imaged using the Cell Voyager 7000 spinning disk confocal microscope (Yokogawa) using the 60× water objective. Primary antibodies used for immunofluorescence were: rabbit anti-ASO (Ionis 13545) (14 (link)), mouse anti-LAMP-1 (BD Bioscience), mouse anti-EEA-1 (BD Bioscience), Cis-Golgi (Abcam), mouse anti-alpha tubulin (DM1A, Cell Signalling) and rat anti-tubulin (Alexa Fluor® 647, Abcam). Secondary antibodies used were donkey anti-rabbit (Alexa Fluor® 488) and goat anti-mouse (Alexa Fluor® 555) sourced from Abcam. Nuclei were stained with Hoechst (Thermo Fisher).

Cells were seeded onto 96-well treated plates (PerkinElmer) at 20% confluence. Cells were left for 24–72 h before incubation with CellMask™ deep red stain (ThermoFisher) and then imaging live or fixing with 4% paraformaldehyde (PFA) and washed three times in PBS. For fixed cell samples, cells were blocked in 2% w/v bovine serum albumin (BSA) in PBS for 30 min before incubation with primary antibodies for 1 h at room temperature. Cells were imaged using the Operetta spinning disk confocal microscope (PerkinElmer) using the 63× water objective. Primary antibodies used for immunofluorescence were mouse anti-LAMP-1 (BD Bioscience), mouse anti-EEA-1 (BD Bioscience), Cis-Golgi (Abcam), mouse anti-alpha tubulin (DM1A, Cell Signalling) and rat anti-tubulin (Alexa Fluor® 647, Abcam). Secondary antibodies used were donkey anti-rabbit (Alexa Fluor® 488) and goat anti-mouse (Alexa Fluor® 555) sourced from Abcam. Nuclei were stained with Hoechst 33342 (Thermo Fisher).