Embed 812 resin kit

Overnight cultures of WT and ΔCbpD in LB were diluted 1:100 and grown in LB to the mid-exponential growth phase. To visualize the effect of serum on bacterial lysis, the bacteria were washed in PBS and suspended in RPMI-HSA supplemented with 10 % (v/v) NHS or HI-NHS for 1 h. The cell pellets were then washed in 0.1 M sodium cacodylate buffer (Sigma) and fixed briefly in a mix of 2% paraformaldehyde (Sigma) and 2.5% glutaraldehyde (Sigma) in 0.1 M sodium cacodylate. Samples were treated with 1% osmium tetroxide (OsO₄; TAAB) before stepwise dehydration in increasing concentrations of ethanol, followed by incubation in acetone. Next, the samples were infiltrated with propylene oxide, embedded using the EMbed 812 resin kit (Electron Microscopy Sciences), and finally polymerized 48 h at 60 °C. Ultrathin sections were collected on copper grids and stained with 2% uranyl acetate and 0.5% lead citrate on an automated contrasting instrument Leica EM AC20 (Leica Microsystems). Finally, the grids were imaged using TEM (Philips CM10 microscope) equipped with a 600-W camera (Gatan) (80–90 kV).

Protist pellets were prepared using the Embed 812 resin kit (Electron Microscopy Sciences (EMS), Hatfield, PA, USA) [55 ]. Briefly, samples were fixed with 4% paraformaldehyde, 1% glutaraldehyde in phosphate buffer (PB, 0.1 M, pH 7.2), followed by 1% OsO4 in PB in the dark. After washing with PB, samples were dehydrated in a 30%–100% gradient ethanol series, infiltrated with increasing amounts of Embed 812 resin in propylene oxide, and cured in molds at 65°C. 80-nm sections were prepared with a Reichert Ultracut E microtome (Leica, Wetzlar, Germany) on 300 mesh copper grids (EMS), and counter stained with saturated 5% uranyl acetate, followed by Reynold’s lead citrate (EMS). Sections were imaged using a Talos L120C transmission electron microscope (Thermo Fisher Scientific, Waltham, MA, USA). See Supplementary Methods for details.

Lactococcus lactis bacteria were transferred to aluminium sample holders and cryoimmobilized immediately using a Leica High-Pressure Machine (HPM 100, Leica Microsystems, Vienna, Austria), and then transferred to liquid nitrogen. Samples were then freeze substituted in a Leica AFS system (Leica Microsystems, Vienna, Austria) with 1% OsO4 in anhydrous acetone with 1% glutaraldehyde, and 1% water at – 90 °C for 1 day, followed by slow warming to room temperature over a period of 7 days. After rinsing in several acetone washes, samples were then gradually infiltrated with mixtures of acetone/epoxy resin and pure epoxy resin (EMbed 812 resin kit, Electron Microscopy Sciences, Hatfield, United States) for 31 h. Samples were embedded in fresh Epon and polymerized at 60 °C for 48 h. Ultrathin Sects. (90 nm) were cut on a Reichert Ultracut E ultramicrotome (Leica, Rueil-Malmaison, France), examined in transmission electron microscope (HITACHI H7800, Japan) operating at 80 kV, and photographed with an AMT nanosprint 43 camera (AMT, Woburn, USA) on the DIMACELL platform (INRAE, Dijon, France).