Fv10 asw laser scanning confocal microscope ver 2.1

Cells treated for the indicated times are fixed in 4% paraformaldehyde in PBS at 1 hour intervals, permeabilized with 0.5% Triton X-100, and blocked with 2% BSA for 30 minutes. The cells are incubated with primary antibody (diluted 1:50) directed against MAP-LC3 or E-cadherin overnight at 4°C. Lysosomal-rich/acidic compartments are visualized with LTR-red (Beyotime, C1046) and incubated at a final concentration of 50 nM for 1 hour. The nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO) for 10 minutes before imaging. An FV10-ASW laser scanning confocal microscope [Ver 2.1] (Olympus Corp, MPE FV1000) is used for co-localization analysis [38 (link)].

Human cardiac fibroblasts were pro-transfected with miR-19a-3p/19b-3p and then treated with 10 ng/ml TGF-β1. MAP-LC3 (green) was labeled with primary anti-MAP-LC3 polyclonal antibody. Goat anti-rabbit IgG/FITC were used as secondary antibody. Cells were simultaneously imaged in the presence of SNLYSO sensor (an autolysosome fluorescent probe, shown red) to visualize autophagy. The nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO) for 10 minutes before imaging. An FV10-ASW laser scanning confocal microscope [Ver 2.1] (Olympus Corp, MPE FV1000) was used for co-localization analysis30 (link).

Cells pretreated with pcDNA3.1 (1 μg) and pcDNA3.1-AEG-1 (1 μg) were incubated with 10 μM DYT-40 and vehicle, respectively, for 24 h. Subsequently, the cells were fixed with 4% paraformaldehyde in PBS at 1-hour intervals, permeabilized with 0.5% Triton X-100, and blocked with 2% BSA for 30 min, followed by incubation with primary antibodies (diluted 1:50) against AEG-1 and NF-κB overnight at 4 °C. Subsequently the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for 10 min prior to imaging. A FV10-ASW laser scanning confocal microscope [Ver 2.1] (Olympus Corp, MPE FV1000) was used for co-localization analysis23 (link).