Plan apo 63 na1.46 oil immersion objective

Cells were grown on poly-L-lysine-coated coverslips (Neuvitro). Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin in PBS. Primary antibodies were applied in blocking buffer supplemented with 0.1% Triton X-100 and incubated overnight at 4°C. Appropriate fluorescent secondary antibodies at a dilution of 1:500 were applied for 60 min at room temperature. Nuclei were counterstained with DAPI before mounting samples with fluorescence-compatible mounting medium (DAKO).
Confocal microscopy was performed at MPIB Imaging Facility (Martinsried, Germany) on a ZEISS (Jena, Germany) LSM780 confocal laser scanning microscope equipped with a ZEISS Plan-APO 63×/NA1.46 oil immersion objective. In case of multi-fluorescence samples, a single-stained control sample was used to adjust emission and detection configuration to minimize spectral bleed-through. Images of cells with inclusions for co-localization studies were subjected to linear unmixing with spectra obtained from the single-stained samples using ZEN software. When fluorescence intensities were directly compared, acquisition settings and processing were kept identical. Images were analyzed with ImageJ (Rasband, W.S., National Institutes of Health, USA) and assembled in Adobe Photoshop CC (Adobe Systems Incorporated, Release 19.1.5).