Computational protocol: ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER

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Protocol publication

[…] Cells were processed for immunofluorescence 24 or 48 h after transfection. Cells grown on glass coverslips were fixed with 4% paraformaldehyde in PBS, pH 7.4, permeabilized with 0.2% Triton X-100, and incubated with primary and secondary antibodies as described previously (; Table S5). 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). Confocal images were obtained using a Leica Biosystems SP8 equipped with an argon laser (488 nm), a DPSS561 laser (561 nm), an HC PL APO 63×/1.3 oil objective, an HC PL APO 100×/1.44 oil objective, and hybrid detectors.Analyses of PO–ER fluorescence overlap were performed using a custom Python implementation of Pearson and Manders colocalization measures, which used the Numpy and Scikit image libraries (, ) In brief, after loading, images were split into red and green channels. Cell regions of interest were manually defined, and Otsu thresholding was used to calculate percentage of overlap of foreground pixels and the Pearson and Manders colocalization measures.For live-cell imaging, fibroblasts were cotransfected with GFP-PTS1 and ACBD5 siRNA 48 h before imaging and plated in 3.5-cm-diameter glass bottom dishes (Cellview; Greiner BioOne). Before image acquisition, a controlled-temperature chamber was set-up on the microscope stage at 37°C, as well as an objective warmer. During image acquisition, cells were kept at 37°C and in CO2-independent medium (Hepes buffered). For fibroblasts, 250 stacks of nine planes (0.5-µm thickness and 100-ms exposure) were taken in a continuous stream. All conditions and laser intensities were kept between experiments. Live-cell imaging data were collected using an IX81 microscope equipped with a CSUX1 spinning disk head (Yokogawa), CoolSNAP HQ2 CCD camera, and 60×/1.35 oil objective. Digital images were taken and processed using VisiView software (Visitron Systems). […]

Pipeline specifications

Software tools MetaMorph, Numpy, scikit-image
Applications Miscellaneous, SPIM, Microscopic phenotype analysis
Organisms Homo sapiens