Lsm 700 laser scan confocal microscope

The experimental steps of immunofluorescence staining were described in detail elsewhere (Hayek et al., 2019 (link)). Briefly, macrophages were seeded on 10 mm sterile coverslips in 24-well plates. After infection and incubation, the cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with ice-cold methanol. The cells were then quenched with 50 mM NH₄Cl in PBS/5% goat serum (GS) followed by incubation with the primary antibody against C. burnetii NMII (Davids Biotechnology). Alexa Fluor 594 (Jackson ImmunoResearch Labs) was used as the secondary antibody. Finally, the slides were mounted with ProLong Diamond containing DAPI (Invitrogen). Immunofluorescent images were taken using the Carl Zeiss LSM 700 Laser Scan Confocal Microscope and the ZEN2009 software.

As in Schäfer et al.^{33 (link)}, cells were seeded on coverslips, washed twice with PBS, fixed with 4% paraformaldehyde (Alfa Aeser) in PBS (Biochrom) for 20 min at room temperature, permeabilized with ice-cold methanol for 30 s, followed by quenching and blocking with 50 mM NH₄Cl (Roth) in PBS/5% goat serum (Life Technologies) for 30 min at room temperature. The coverslips were incubated with primary antibodies directed against Flag M2 (Sigma Aldrich) and C. burnetii diluted in PBS/5% goat serum for 1 h at room temperature, washed three times with PBS and further incubated with secondary Alexa Fluor labelled antibodies Alexa 488 and Alexa 594 (Dianova) diluted in PBS/5% goat serum for 1 h. After three washes with PBS, the cells were mounted using ProLong Diamond with DAPI to visualize cell nuclei and bacterial DNA. Confocal fluorescence microscopy was performed using a Carl Zeiss LSM 700 Laser Scan Confocal Microscope.

Hemocytes from 3 Galleria mellonella larvae infected with either C. burnetii, C. burnetii ΔdotA, C. burnetii pFlag-AnkG or C. burnetii pFlag-AnkF were collected at 1 day and 5 days post infection^{38 (link)}. Cells were seeded and centrifuged on poly-l-lysine-coated coverslips, washed twice with PBS, fixed with 4% paraformaldehyde (Alfa Aeser) in PBS (Biochrom) for 20 min at room temperature, permeabilized with 0.1% Triton- X 100, followed by quenching and blocking with 50 mM NH₄Cl (Roth) in PBS/5% goat serum (Life Technologies) for 30 min at room temperature. The coverslips were incubated with primary antibodies directed against C. burnetii and actin (phalloidin labeled with Alexa Fluor 674) diluted in PBS/5% goat serum for 20 min at room temperature, washed three times with PBS and further incubated with secondary Alexa Fluor labelled antibodies Alexa 488 diluted in PBS/5% goat serum for 20 min. After three washes with PBS, the cells were mounted using ProLong Diamond with DAPI to visualize cell nuclei and bacterial DNA. Confocal fluorescence microscopy was performed using a Carl Zeiss LSM 700 Laser Scan Confocal Microscope. In Z-Stack images with 1 µm distance 100 randomized cells were counted and determined whether they were infected or not. The experiment was performed three times.