Flouromount

HeLa cells were grown to confluency on a surface-treated coverslip placed in a sterile 24-well tissue culture plate wherein DMEM supplemented with 5% fetal bovine serum was added. Media was removed from the wells and cells were washed thrice with 1 × PBS to remove the residual antibiotics and this was followed by infection. Bacterial cells were grown overnight in LB at 37 °C and used to infect HeLa cells at an MOI of 1:5. Following infection, the Hela cells were incubated at 37 °C for 15 min, 30 min and 1 h. Media was removed from the well and the coverslip with adherent cells was washed thrice with 1 × PBS and then fixed in 3.7% formaldehyde for 20 min at room temperature (28 °C) on a rocker. This was followed by washing with 1× PBS thrice for 5 min and staining with Phalloidin-iFlour 488 Conjugate (AAT Bioquest, Sunnyvale, CA, USA) (1×) in 1 × PBS (1% BSA) for 2 h at room temperature (28 °C) in a rocker. After 2 h, phalloidin solution was removed and cells were washed thrice with 1 × PBS. Nuclear staining was performed by incubating the cells with 300 nM 4′,6-diamidino-2-phenylindole (DAPI) for 30 min at room temperature in a rocker. Cells were washed thrice in 1 × PBS and the coverslip mounted on the slide using flouromount (Sigma-Aldrich, St. Louis, MI, USA). Images were captured using a fluorescence microscope (Olympus-BX53).

For initial IHC, tissue sections were blocked in Ultravision Block (Thermo scientific) for 1 h. Incubation with primary antibody (Table 1) in a total volume of 100 μL Ultra Vision Block with 10% goat serum (Sigma Aldrich) was performed overnight at 4 °C in a humidity box. Incubation with fluorescent secondary antibodies (Table 2) were performed for 30 min in 100 μL Ultra Vision Block with 10% goat serum. Flouromount (Sigma-Aldrich) was used for mounting. A frozen tissue microarray with 5 μm sections (BioChain^®, Cat. T6235086-1) was blocked and stained as described above. Fluorescence microscopy was performed with a Leica DMI 3000 B microscope with Cell B imaging software.

NCI-H292 epithelial cells were grown on plastic cover slips (Thermanox) placed in 24-well plates (Costar). After 3h infection at MOI 20, cell culture medium containing bacterial cells was discarded, adherent cells were washed twice with 1x Dulbecco’s PBS (without Ca²⁺ and Mg²⁺, Sigma Aldrich), and cover slips were mounted on 10μl Flouromount (Sigma Aldrich) drops on glass slides. Edges of the cover slips were sealed for long-term storage. Samples were imaged under the LWD 20x/0.40 objective of the Nikon Eclipse 80i microscope with a DS-U1 camera using the ACT-2U program with the following settings: exposure control: manual, AE: M, Gain: 1, DF/FL, color effect: Mono.