Zen system imaging software

Escherichia coli intact biofilms were visualized using an inverted Zeiss Axio Observer Laser Scanning Microscope (LSM) 880 equipped with a C-APOCHROMAT 40x/1.2 Water Corr-UV-VIS-IR objective. The following lasers were used: 458 nm, 488 nm, 514 nm Argon laser and 561 nm DPSS laser. Each experiment was performed at least three times on independent biological replicates. Obtained images and z-stack projections were visualized using Zeiss ZEN System imaging software (Zeiss). Thickness of pellicles was measured using an upright Leica TCS SP5 confocal laser scanning microscope equipped with HC PL FLUOTAR 10x/0.3 dry objective.

Microscopy was performed on a LSM 880 confocal microscope (Carl Zeiss, Oberkochen, Germany). Between 2 or 3 days after transfection, cells were incubated with mAb C7 (Jacobson et al. 1999) for 1 h at room temperature, which binds the extracellular domain of the AChR δ‐subunit, diluted 1:1000 in staining medium (DMEM containing 20 mm Hepes and 1% BSA), before being washed three times with staining medium. Cells were fixed with 3% paraformaldehyde at room temperature for 10 min, washed three times with PBS and incubated with secondary antibody Alexa Fluor® 594 goat anti‐mouse IgG (H+L) (catalogue no. A11005; Invitrogen; RRID:AB_141372) diluted 1:750 in staining medium. Cells were washed three times in PBS and mounted in fluorescence mounting medium (Dako Cytomation, Glostrup, Denmark). Images were captured using the ZEN System imaging software (Carl Zeiss).