Spectral lsm710 microscope

For confocal microscopy Dictyostelium cells were transferred to Ibidi μ-Slide-eight-well chambers (Ibidi, 80,826) and directly observed in vivo (for cells expressing fluorescently-tagged proteins) or fixed for immunofluorescence detection. Fixation was performed for 15 min with 2% paraformaldehyde (Merck, 104,005) dissolved in 2x PBS (266 mM NaCl, 16 mM Na₂HPO₄, 4 mM KH₂PO₄, pH 7.4). After three washes with 1x PBS (133 mM NaCl, 8 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4) cells were blocked (blocking solution: 2% BSA [Sigma-Aldrich, A3059-10 G]; 0.5% NP-40 [Sigma-Aldrich, 74,385]; 1x PBS) for 30 min. Primary antibody incubation was left overnight at 4°C in blocking solution. The Atg8 antibody was kindly provided by Jason King (Department of Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom) and used at 1:2000; the FLAG antibody (Cell Signaling Technology, 8146) was used at 1:1000. After three washes with 1x PBS cells were incubated 1 h at room temperature with the indicated secondary antibody conjugated to Alexa Fluorophores (Molecular Probes, A11034, A11035, A11029, A11030) in blocking solution at 1:500. Confocal images were acquired with an inverted Zeiss spectral LSM710 microscope.

Immunofluorescence analysis was performed as described14 (link)69 (link) on cells grown on coverslips, fixed in paraformaldehyde and permeabilized with 0.05% Triton-X100. Cells were incubated for 2 h at 37 °C in a humidified chamber with the primary and secondary antibodies, described in Supplementary Table 4. Phalloidin-647 (Amersham) was used for F-actin stain. Confocal microscopy analyses were performed using a Zeiss Spectral LSM710 microscope (x40 oil objective) and Zen2009 software.