10 nm colloidal gold particles

After growing overnight in DMEM at 37°C, bacterial cultures were centrifuged, washed with PBS and fixed with 4% formaldehyde. After fixation, preparations were washed, blocked with 0.2% BSA in PBS, and incubated overnight with rabbit anti-EspA antibody (1:10 dilution in PBS) at 4°C. Preparations were subsequently washed with PBS and incubated with goat anti-rabbit antibody labeled with 10 nm colloidal gold particles (Sigma-Aldrich) diluted 1:10 in PBS, for 3 h at room temperature. After further washings, preparations were negatively stained (or not) with 2% uranyl acetate in water, in order to facilitate counting and measurement of the EspA filaments and placed onto Formvar-coated nickel grids. After air-dried, preparations were then analyzed under transmission electron microscopy (TEM) (LEO 906E – Zeiss, Germany) at 80 kV.

Cell pellets in cryoprotectant solution were loaded onto membrane carriers (Mager Scientific) and cryo-fixed using the EM PACT2 high-pressure freezer (Leica). Cryo-fixed cells were processed by freeze substitution using an AFS2 automated freeze substitution unit (Leica). Briefly, frozen pellets were incubated at −90°C in a solution of tannic acid in acetone, followed by slowly warming to room temperature in a solution of uranyl acetate and osmium tetroxide in acetone. Samples were infiltrated with Durcupan ACM resin (Sigma-Aldrich), and blocks were polymerized at 60°C. Thin sections (60–90 nm) were cut using the UC7 ultramicrotome (Leica), post-stained with 2% aqueous uranyl acetate and Sato’s lead, and imaged on a Tecnai T12 microscope (FEI) operating at 80 kV. Thicker 250-nm sections were screened on a 200 kV (CM200 Philips) microscope. Samples from all time points were screened in both thin and thick sections. For tomography, serial sections of 250-nm thickness were collected on LUXFilm-coated 2 × 1 mm copper slot grids (Luxel), post-stained, carbon-coated, and overlaid with 10-nm colloidal gold particles (Sigma-Aldrich) for use as fiducial markers. At least 50 sections were screened for morphological determination from two representative resin blocks for each time point and condition.