Vancomycin

Bacterial strains 10 and 10ΔmapZ were prepared as described in 2.3 and grown until an OD₆₀₀ of approximately 0.2. Then 1 ml of the bacterial culture was transferred into a sterile 1.5 ml Eppendorf tube and 5 µl vancomycin‐FL (stock: 100 µg/ml; Thermo Fisher Scientific) as well as 5 µl vancomycin (stock 100 µg/ml; Carl Roth) were added. The cultures were vortexed and incubated at 37°C for additional 20 min. Afterward, the cultures were centrifuged for 10 min at 1690 × g at 4°C. The pellets were washed with HBSS buffer (Thermo Fisher Scientific), resuspended in PBS buffer (Sigma‐Aldrich), and fixed in PBS buffer containing 3% formaldehyde. Bacteria were transferred to poly‐l‐lysine (Sigma‐Aldrich) coated coverslips, mounted with ProLong^® Gold Antifade Reagent (Cell Signaling Technology), and stored at 4°C until examination.
Confocal microscopy was performed using a TCS SP5 confocal laser scanning microscope equipped with a 63× 1.40–0.60‐NA oil HCX Plan Apochromat objective (Leica). Image stacks with a z‐distance of 0.13 µm per plane were acquired using a 1‐Airy‐unit pinhole diameter. Images were deconvolved using Huygens^® Essential 20.10 (Scientific Volume Imaging). Maximum intensity projections of 3D‐stacks were generated and adjusted identically for brightness and contrast in ImageJ/Fiji (Schindelin et al., 2012 (link)).