Anti mouse c5b 9

Immunofluorescence microscopy was performed as previously described^{125 (link)}, with modifications. Bacteria were prepared as described above under “Bacterial Survival Assays in Blood” and incubated with 10% (v/v) NHS or HI-NHS for 45 min at 4 °C under shaking. The samples were washed twice with PBS supplemented with 1% BSA (Sigma) (PBS/BSA). Next, the bacteria were fixed by PBS/BSA containing 4% paraformaldehyde (PFA, Alfa Aesar) for 20 min at RT. Bacteria were washed once with PBS/BSA, blocked in PBS/BSA for 1 h at RT, and incubated with mouse anti-C5b-9 (1 μg ml⁻¹, aE11, Santa Cruz) followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG (1 μg ml⁻¹, Life Technologies) for 45 min. Bacteria were washed once with PBS/BSA and then with Hanks Balanced Salt Solution (HBSS, Gibco). Ultimately, the pellet was resuspended in HBSS supplemented with the lipophilic dye FM5‐95 (Invitrogen, 10 μg ml⁻¹) to label bacterial membranes. Labeled bacterial cells were mounted onto glass slides (covered with a thin layer of agarose gel) and secured with coverslips. Fluorescence microscopy was performed on a Zeiss AxioObserver equipped with an ORCA‐Flash4.0 V2 Digital CMOS camera (Hamamatsu Photonics) and ZEN Blue software. Images were acquired through a ×100 phase-contrast objective and analyzed by ImageJ/Fiji (v 2.1.0/1.53c).