Ec plan neofluar 100 1.3 oil

Cells (2 × 10² cells/well) were plated in a 6-well-plate containing coverslips in 2 ml of culture media and cultured overnight. The cells were washed twice with PBS and then fixed with 4% formaldehyde in PBS at room temperature (RT) for 30 min. The fixed cells were washed thrice with PBS for 5 min. After washing, the cells were permeabilised with 0.1% TritonX-100 in PBS for 15 min, followed by three washes with PBS. The fixed cells were blocked with 3% (w/v) BSA, stained with an anti-Vinculin antibody (Abcam, CA, Cat No. ab129002) and rhodamine phalloidin (R415, Invitrogen, USA) overnight at 4 ℃ in the dark, followed by incubation with Fluor488-conjugated secondary antibodies (Invitrogen, CA, Cat No. A21202) after three washes with PBS. Each coverslip was mounted with a medium containing the nuclear stain DAPI (Vectashield) (H 1200, Vector Laboratories, USA). The fluorescence images were obtained with a Laser Scanning Confocal Microscope (LSM 780, ZEISS, Germany) and visualised with the 100× oil immersion objective lenses (EC Plan-Neofluar 100×/1.3 oil).

The viability of E. coli ATCC 25922, S. aureus ATCC 29213, and P. aeruginosa ATCC 27853 in the aggregates was assessed by using the LIVE/DEAD® BacLight^TM Bacterial Viability kit (Invitrogen, Molecular Probes, Carlsbad, CA). For this purpose, the bacterial suspension was prepared as described above for VCA. Bacteria were then treated by 5 μm rTCP96 or 5 μm GKY25 in 10 mm Tris, pH 7.4. The buffer was used for control. After 2 h, the samples were mixed with 3 μl of the dye mixture for each ml of the bacterial suspension, as reported on the manufacturer's protocol, and incubated in the dark at room temperature for 15 min. At the end of incubation, 5 μl of the stained bacterial suspension were trapped between a slide and an 18-mm square coverslip.
We examined 10 view fields (1 × 1 mm) of the mounted samples from three independent sample preparations using a Zeiss AxioScope A.1 fluorescence microscope (objectives: Zeiss EC Plan-Neofluar ×100/1.3 oil; camera: Zeiss AxioCam MRm; acquisition software: Zeiss Zen 2.6 (blue edition). For the analysis we measured the area of all aggregates possible to distinguish in each picture (43 for E. coli, 42 for P. aeruginosa, and 67 for S. aureus).