Coolsnap hq2 ccd camera

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).

Cells were processed for immunofluorescence 24 h after transfection as described previously.⁴¹ (link) Cell imaging was performed using an Olympus IX81 microscope equipped with an UPlanSApo 100x/1.40 Oil objective (Olympus Optical, Hamburg, Germany), eGFP ET filter-set (470/40 ET Bandpass filter, Beamsplitter T495 LPXR and 525/50 ET Bandpass filter (Chroma Technology GmbH, Olching, Germany)), and TxRed HC Filter Set (562/40 BrightLine HC Beamsplitter HC BS 593, 624/40 BrightLine HC (Semrock, Rochester, USA)). Digital images were taken with a CoolSNAP HQ2 CCD camera and adjusted for contrast and brightness using MetaMorph 7 (Molecular Devices, https://www.moleculardevices.com/systems/metamorph-research-imaging/metamorph-microscopy-automation-and-image-analysis-software). Confocal images were obtained using a Leica SP8 equipped with: Argon laser (488), DPSS561 laser (561), HC PL APO 63x/1.3 Oil objective, HC PL APO 100x/1.44 Oil objective, Hybrid detectors (HyD).