Three-dimensional structured illumination microscopy with enhanced axial resolution - NIBIBgov
and grown for 4 hours at 37 °C, shaking at 250 r.p.m. Before imaging, 1 ml of culture was removed and centrifuged at 14,000For some experiments, we stained bacterial membranes. Bacteria were diluted in 1 ml of PBS and stained with CellBrite Fix 488 or CellBrite Fix 555 for 5 minutes at room temperature at 1× working concentration according to the manufacturer’s guidelines. Stained bacteria were washed three times with 1× PBS, each time centrifuging the solution at 3,000 r.p.m.
To immunolabel microtubules, U2OS cells were treated with pre-extraction buffer at 37 °C for 30 seconds, and then the buffer was quickly replaced with microtubule fixation buffer at 37 °C for 15 minutes. Fixed cells were rinsed three times with 1 ml of 1× PBS , quenched in 1 ml of 0.1% sodium borohydride/PBS solution for 7 minutes at room temperature and blocked in 100% FBS at 37 °C for 1 hour.
For immunolabeling Tomm20, U2OS cells were fixed with 2% paraformaldehyde and 0.125% glutaraldehyde in 1× PBS for 15 minutes at room temperature. Cells were rinsed three times with 1× PBS, permeabilized by 0.
We stained mitochondria and internal membranes with synthetic dyes for live cell imaging. In the former case, U2OS cells were incubated with MitoTracker Green FM for 15 minutes at 37 °C and rinsed three times with 1× PBS immediately before imaging. In the latter case, U2OS cells were incubated in DMEM media with Potomac Gold for 30 minutes at 37 °C. Immediately before imaging, cells were rinsed three times with 1× PBS.
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