Lsm 880 clsm

MDBK cells grown on glass coverslips were transfected as described above with the expression plasmids encoding the UL30-HA and UL42- FLAG proteins with or without the drug IVM. At 24 h post transfection, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (136.9 mM NaCl, 2.7 mM KCl, 7.0 mM Na₃PO4, 0.9 mM Na₃PO₄ at pH 7.4) at 37°C for 20 min and permeabilized with 0.2% Triton X-100 in PBS for 10 min. Cells were subsequently incubated in blocking buffer (1% bovine serum albumin (BSA) (Biosharp, Hefei, China) and 0.1% Tween 20 in PBS) for 2 h. Cells were subsequently incubated with the primary antibodies including mouse anti-HA and rabbit anti-FLAG (Beyotime, Haimen, China) at 1:500 dilution in blocking buffer. Finally, cells were stained with secondary antibodies, including Cy3-conjugated goat anti-mouse and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit (Beyotime, Haimen, China) at 1:1000 dilutions in blocking buffer. The DNA was counterstained with 4, 6-diamidino-2-phenylindole (DAPI). After each step, cells were washed three times with 1× PBS. The fluorescent signals were observed using Zeiss LSM 880 CLSM (Carl Zeiss, Jena, Germany), equipped with a 63x objective. Images were processed using Image J (NIH) as described previously [25 (link)].

The SCFM2 culture method described above was used for imaging. Three wells (S. aureus monoculture, P. aeruginosa monoculture, and coculture) per optical chamber were used for each replicate imaging experiment. Each experiment imaged a single position in each well, once per hour. All images were acquired with a Zeiss LSM 880 CLSM utilizing Zen image capture software. Detection of DsRed-expressing S. aureus cells was performed with an excitation wavelength centered at 587 nm and an emission wavelength centered at 610 nm. Detection of GFP-expressing cells was performed using an excitation wavelength centered at 488 nm and an emission wavelength centered at 509 nm. All images were acquired using a 63× oil-immersion objective. All data were stored as 1,024- by 1,024-pixel slices in stacks of 91 8-bit images. Each voxel is 0.264 by 0.264 by 0.44 μm³.