Lsm 510 laser scanning fluorescence confocal microscope

Stress-induced cells were seeded onto glass coverslips (Marienfeld Laboratory Glassware) at 4 x 10⁵ cells/ml in six-well plates and fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature. Fixed cells were washed with PBS and permeabilized with 0.1% Triton X-100 in PBS for 5 minutes. After washing with PBS, the coverslips were incubated with BAG5 (Santa Cruz, USA)- and α-synuclein (Genetex, USA)-specific antibodies overnight at 4°C. The coverslips were incubated with Fluorescein (FITC) AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch, USA) and Rhodamine Red-X-conjugated goat-anti-rabbit IgG (H+L) (Jackson ImmunoResearch, USA) overnight at 4°C. After washing twice with PBS, the coverslips were stained with DAPI for 10 minutes, and cells were mounted with mounting medium (Sigma-Aldrich, USA). Confocal images were captured under a Zeiss LSM 510 Laser Scanning Fluorescence Confocal Microscope. The percentage of cells with colocalization was determined by counting yellow blobs of at least 1,000 cells per strain using ImageJ software. Immunofluorescence images were captured under a Zeiss AxioImager M1. The percentage of cells with α-synuclein foci was determined by counting at least 1,000 cells per strain using ImageJ software.

MSTO-211H cells were seeded into eight-chamber culture slides (BD Falcon). The next day, cells were rinsed with ice-cold PBS buffer and fixed with 4% paraformaldehyde for 10′ at room temperature and then permeabilized with 1% Triton X-100. Cells were incubated overnight with the indicated antibody. The day after, cells were washed with cold PBS three times for 3 min each and stained for 2 h with a secondary antibody Alexa 488-conjugated goat anti-mouse IgG or anti-rabbit IgG (Molecular Probes Cells) and counterstained with DAPI (40,6-diamidino-2-phenylindole dihydrochloride). Cells were examined under a Zeiss LSM 510 laser scanning fluorescence confocal microscope (Zeiss, Wetzlar, Germany).