Ctr 7000 hs controller

NRK-49F fibroblasts were seeded on coverslips to a confluence of 50–60% in 24-well plates and infected with wild-type, ∆mrdA and ∆PBP2_SAL strains. Infected cells were fixed at 2, 4, 8 and 24 hpi in 3% PFA (15 min, RT), and processed for immunofluorescence microscopy, as described^{70 (link)}. Briefly, after PFA fixation, the infected cells were washed in PBS pH 7.4 and incubated for 10 min at RT in blocking solution containing 0.1% (w/v) saponin and 1% (v/v) goat serum. Incubations with primary and secondary antibodies were performed in this same solution of 0.1% (w/v) saponin and 1% (v/v) goat serum during 45 min each, with three washes in PBS pH 7.4 after the respective incubations. Coverslips were finally mounted on slides using ProLong Gold Antifade (Molecular Probes). Rabbit polyclonal anti-S. Typhimurium LPS (cat. no. 229481, 1:1000, Difco Antiserum-BD Diagnostics, Sparks, MD) and goat polyclonal anti-rabbit IgG conjugated to Alexa 488 (1:1000, cat. no. A-11008, ThermoFisher Scientific), were used as primary secondary antibodies, respectively. Images were acquired on an inverted Leica DMI 6000B fluorescence microscope with an automated CTR/7000 HS controller (Leica Microsystems) and an Orca-R2 charge-coupled-device (CCD) camera (Hamamatsu Photonics).

At the desired growth conditions, bacteria were fixed with 3% (w/v) paraformaldehyde (PFA) for 10 min and adjusted to a final PFA concentration of 1%. These fixed bacteria were centrifuged (4300  × g, 2 min, RT) and resuspended in phosphate-buffered saline (PBS), pH 7.4. A volume of 50 μL of the bacterial suspension was dropped on poly l lysine-precoated coverslips and incubated for 15 min at RT. The attached bacteria were washed three times with PBS, and the coverslip was mounted on slides using ProLong Gold Antifade (Molecular Probes). Images were acquired on an inverted Leica DMI 6000B microscope with an automated CTR/7000 HS controller (Leica Microsystems) and an Orca-R2 charge-coupled-device (CCD) camera (Hamamatsu Photonics).

NRK-49F fibroblasts were seeded on coverslips to a confluence of 50–60% in 24-well plates and infected with wild-type, ∆mrdA and ∆PBP2_SAL strains. Infected cells were fixed at 2, 4, 8 and 24 hpi in 3% PFA (15 min, RT), and processed for immunofluorescence microscopy, as described^{70 (link)}. Briefly, after PFA fixation, the infected cells were washed in PBS pH 7.4 and incubated for 10 min at RT in blocking solution containing 0.1% (w/v) saponin and 1% (v/v) goat serum. Incubations with primary and secondary antibodies were performed in this same solution of 0.1% (w/v) saponin and 1% (v/v) goat serum during 45 min each, with three washes in PBS pH 7.4 after the respective incubations. Coverslips were finally mounted on slides using ProLong Gold Antifade (Molecular Probes). Rabbit polyclonal anti-S. Typhimurium LPS (cat. no. 229481, 1:1000, Difco Antiserum-BD Diagnostics, Sparks, MD) and goat polyclonal anti-rabbit IgG conjugated to Alexa 488 (1:1000, cat. no. A-11008, ThermoFisher Scientific), were used as primary secondary antibodies, respectively. Images were acquired on an inverted Leica DMI 6000B fluorescence microscope with an automated CTR/7000 HS controller (Leica Microsystems) and an Orca-R2 charge-coupled-device (CCD) camera (Hamamatsu Photonics).