4 oil dic m27 plan apochromat objective

For immunofluorescence microscopy, cells were seeded in µ-Slide 8 Well (Ibidi, Graefeling, Germany, #80826). Cells were treated with 100 nM Lysotracker^TM Deep Red (Thermo Fisher Scientific, Waltham, MA, USA, #L12492). After the incubation time, the treatment medium was removed and replaced by DMEM without phenol red (Thermo Fisher Scientific, Waltham, MA, USA, #31053028). Representative images were acquired with an Axio Observer 7 fluorescence microscope (Carl Zeiss Microscopy, Jena, Germany) using a 40x/1.4 Oil DIC M27 Plan-Apochromat objective (Carl Zeiss Microscopy, Jena, Germany) and an ApoTome 2 (Carl Zeiss Microscopy, Jena, Germany).

For BFA washout assays, HeLa wt cells were seeded on glass coverslips in 24-well plates. On the next day, cells were treated with 0.1% DMSO, 10 nM prodigiosin or 100 nM prodigiosin. After 24 h, the treatment medium was removed, cells were washed once with DPBS and then treated with 5 µg/mL BFA or 0.1% DMSO as a control for 2 h. Cells were then washed with DPBS four times and incubated with fresh culture medium for 0/15/30/45/60/120 min. After the respective BFA washout time, cells were fixed in ice-cold methanol for 15 min on ice, washed three times with DPBS and blocked in 3% BSA (Roth, Karlsruhe, Germany, #8076) overnight. Samples were incubated with primary antibodies diluted in 3% BSA for 2 h and then washed three times with DPBS, incubated with the appropriate secondary antibodies diluted in 3% BSA for 30 min and washed three times with DPBS. Afterwards, cells were embedded in ProLong Glass Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA, #P36980) containing 1 µg/mL DAPI (Roth, Karlsruhe, Germany, #6335.1). Images were recorded with an Axio Observer 7 fluorescence microscope (Carl Zeiss Microscopy, Oberkochen, Germany) using a 40x/1,4 Oil DIC M27 Plan-Apochromat objective (Carl Zeiss Microscopy, Oberkochen, Germany) and an ApoTome 2 (Carl Zeiss Microscopy, Oberkochen, Germany).

For immunofluorescence microscopy, HeLa cells were seeded on glass coverslips in 24-well plates. After treatment, cells were fixed in 4% paraformaldehyde for 15 min on ice, quenched with 50 mM NH₄Cl for 15 min and permeabilized with 50 µg/mL digitonin (Sigma-Aldrich, St. Louis, MO, USA, #D141) for 5 min. Fixed samples were blocked with 3% BSA (Roth, Karlsruhe, Germany, #8076) for 30 min or overnight and incubated with primary antibodies diluted in 3% BSA for 2 h. Samples were then washed three times with DPBS, incubated with the appropriate secondary antibodies diluted in 3% BSA for 30 min and washed three times with DPBS. Afterwards, cells were embedded in ProLong Glass Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA, #P36980) containing 1 µg/mL DAPI (Roth, Karlsruhe, Germany, #6335.1). Images were recorded with an Axio Observer 7 fluorescence microscope (Carl Zeiss Microscopy, Oberkochen, Germany) using a 40x/1,4 Oil DIC M27 Plan-Apochromat objective (Carl Zeiss Microscopy, Oberkochen, Germany) and an ApoTome 2 (Carl Zeiss Microscopy, Oberkochen, Germany).