1.4 na objective lens

Other primary antibodies against the following proteins were used in immunofluorescence experiments: AURKB (Novus, Cat# NBP2-50039, RRID:AB_2895237), and p-Smad2 (Cell Signaling Technology Cat# 3108, RRID:AB_490941). Secondary antibodies were: donkey anti-rabbit Alexa Fluor 555 (Thermo Fisher Scientific Cat# A-31572, RRID:AB_162543), donkey anti-mouse Alexa Fluor 555 (Thermo Fisher Scientific Cat# A-31570, RRID:AB_2536180), goat anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific Cat# A-11029, RRID:AB_2534088), and goat anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific Cat# A32731, RRID:AB_2633280). Immunofluorescence assays were performed as described previously.¹⁶ (link) Briefly, cells were plated on coverslips, fixed in 4% paraformaldehyde for 30 min, and then treated with 0.2% Triton X-100 in PBS for 5 min and blocked with 10 mM glycine. Incubation with primary antibodies was performed for 1 h at room temperature, followed by washing in PBS and incubation with secondary antibodies. Photomicrographs were obtained using a confocal microscope LSM 710 (Carl Zeiss) with a 63 × /1.4 NA objective lens (Carl Zeiss). The images were acquired under oil immersion at room temperature, using Zen 2011 software.

Immunofluorescence assays were performed as described previously [12 (link), 47 , 48 ]. In brief, cells were plated on coverslips and fixed in 4% paraformaldehyde for 30 min, treated with 0.2% Triton X-100 in PBS for 5 min at room temperature and then blocked with 10 mM glycine. Incubation with the primary antibodies was performed for 1 h at room temperature, followed by washing in PBS and then incubation with the secondary antibodies. Photomicrographs were obtained using a confocal microscope LSM 710 (Carl Zeiss) using a 63 × 1.4/NA objective lens (Carl Zeiss). The images were acquired using the Zen 2010 software in the presence of immersion oil at room temperature.

For live imaging or fixed sample preparation of bacteria inside L929 host cells, L929 cells were grown directly on chambered coverslip slides (Ibidi, USA). These were infected with labelled or unlabelled bacteria and either imaged live or fixed and processed for labelling with fluorescently-conjugated antibodies. Samples were fixed with 4% paraformaldehyde for 10 min at room temperature, and then washed three times with PBS. Samples were permeabilised using 0.5% triton X on ice for ten minutes, and then washed three times with PBS. Immunofluorescent labelling was performed using a rat monoclonal antibody against the TSA56 gene diluted to 1/200, followed by an alexa 488-conjugated secondary anti-rat antibody diluted to 1/1000 (ThermoFisher Scientific, UK). Incubations were performed for 30–60 min at room temperature, and washed three times with PBS in between. Samples were mounted using hardset mounting media (Vectashield, Vector Laboratories, USA). Imaging was performed using a Zeiss LSM 7000 equipped with a 63 × 1.4 NA objective lens (Carl Zeiss, USA).