18mm diameter cover glass

Cells were grown on 18mm diameter cover glass (Marienfeld). After 48hr incubation, the cells were rinsed twice with 1X PBS and fixed and permeabilized in methanol-acetone mixture (1:1) for 7min at −20°C. Fixed cells were blocked with 5% BSA/PBS-T (PBS, 0.2% Tween-20) for 1hr at room temperature, and then incubated with CSF-1R antibody (1:250 dilution) and TSC-22 antibody (1:500 dilution) for 16hrs at 4°C. The cells were washed three times with 1X PBS for 5min each and incubated with alexa fluor 488 goat anti-rabbit IgG (Green) and alexa fluor 568 goat anti-mouse IgG (Red) antibody (Invitrogen, 250:1 dilution) in darkness for 1hr at room temperature. Finally, the cells were counterstained with 1μg/ml DAPI for 1min at room temperature and mounted on slides. The signals and co-localization were detected using the confocal fluorescence microscopy

Cells were grown on 18mm diameter cover glass (Marienfeld). After 48hr incubation, the cells were rinsed twice with 1X PBS and fixed and permeabilized in methanol-acetone mixture (1:1) for 7min at −20°C. Fixed cells were blocked with 5% BSA/PBS-T (PBS, 0.2% Tween-20) for 1hr at room temperature, and then incubated with CLU polyclonal antibody (1:300 dilution) and eIF3f polyclonal antibody (1:300 dilution) for overnight at 4°C. The cells were washed three times with 1X PBS for 5min each and incubated with alexa fluor 488 goat anti-rabbit IgG (Green) and alexa fluor 568 donkey anti-goat IgG (Red) antibody (Invitrogen, 1000:1 dilution) in darkness for 1hr at room temperature. Finally, the cells were counterstained with 1μg/ml DAPI for 1min at room temperature and mounted on slides. The signals and co-localization were detected using the confocal fluorescene microscopy.