Immunofluorescence Microscopy of PEX11β

Cells were processed for immunofluorescence 24 h after transfection (if not indicated otherwise). Cells grown on glass coverslips were fixed with 4% paraformaldehyde in PBS, pH 7.4. For detection of PEX11β, cells were permeabilised with 2.5 μg/ml digitonin, as PEX11β is extracted from peroxisome membranes after post-fixation Triton X-100 treatment (Bonekamp et al., 2013a (link); Schrader et al., 2012 (link)). For other detections or differential permeabilisation, cells were either permeabilised with 0.2% Triton X-100 or 2.5 μg/ml digitonin. Cells were incubated with primary and secondary antibodies as described previously (Bonekamp et al., 2013b ) (Table S4). Cell imaging was performed using an IX81 microscope (Olympus) equipped with an UPlanSApo 100×/1.40 oil objective (Olympus). Digital images were taken with a CoolSNAP HQ2 CCD camera and adjusted for contrast and brightness using the Olympus Soft Imaging Viewer software and MetaMorph 7 (Molecular Devices).