Em menu software

The serial sections were imaged using a TECNAI T12 Biotwin transmission electron microscope (Thermo Fisher Scientific, Waltham, MA) operated at 120 kV and equipped with an F214 charge-coupled device (CCD) camera (TVIPS GmbH, Germany). Images of whole cells in metaphase were acquired at 1200× magnification using EMMenu Software (TVIPS GmbH, Germany). To choose a cell for electron tomography, the metaphase plate had to be correctly formed when looking at the chromosome area in 3D and for each chosen cell the half-spindle and the MT angle were measured in the 3D volumes as described (see the Light microscopy section). For this, the EM stacks were projected in 3D. The chromosome and microtubule area were estimated by manually labeling the chromosome and MT area. Both the half-spindle angle and MT angle were finally calculated using the ZIB Amira software (Zuse Institute Berlin, Germany).

1-day-old MyD88^+/+ and MyD88^-/- mice were infected with wild type S. Typhimurium and sacrificed at day 4 p.i.. Tissue samples were prepared for ultrastructural analysis as previously described [88 (link)]. After embedding, samples were post-fixed with 1% osmium tetroxide and contrasted with 2% uranyl acetate, both for 2 h. Samples were dehydrated with a graded ethanol series, followed by infiltration with epoxy resin and overnight heat polymerization. Thin, 70 nm sections were prepared using an ultramicrotome Ultracut UCT (Leica Microsystems) and contrasted with 0.2% lead citrate. Sections were analyzed with a JEM-1400 TEM microscope (Jeol) and images were recorded with TemCam-F216 camera using EM MENU software (both Tvips).

Negative stain grids were prepared as described under immunogold labeling. Micrographs were collected manually at 120 kV using a Tecnai G2 Spirit TWIN electron microscope with a defocus value of 0.5–2.0 μm. Images were collected using a Tietz TemCam-F416 CMOS camera at a nominal magnification of ×67,000 and a pixel size of 1.57 Å employing the EM-Menu software (TVIPS GmbH). Data processing was done using the SPRING suite employing CTFFIND, CTFTILT, EMAN2, and SPARX (39 (link)– (link)43 (link)). Straight regions of the pilus filaments were extracted, segmented, and averaged to determine outer dimensions and helical parameters. Averaged intensity width profiles were plotted, and the outer diameter was taken as the distance between the two outer minima in the intensity profile. Power spectra were calculated from the averages, and the most prominent layer lines were identified. The pitch was determined in real space from intensity normal profiles along the helical axis as well as from Fourier space based on layer line positions.

293T cells expressing APEX2-tagged TECPR2 WT and L440Rfs were grown on aclar sheets (Science Services) and fixed with 2.5% glutaraldehyde (EM-grade, Science Services) in 0.1 M pH 7.4 sodium cacodylate buffer (CB) for 30 min on ice. Endogenous peroxidases were blocked with 20 mM glycine (Sigma) for 5 min on ice and cells washed in CB. Cells were saturated with freshly prepared 1× diaminobenzidine (DAB, in CB supplemented with 2 mM calcium chloride) for 5 min and APEX2 activity was triggered with 1× DAB supplemented with 10 mM H₂O₂ (Sigma) for 40 min on ice. Cells were washed with CB and subsequently postfixed and contrasted in reduced osmium (1.15% osmium tetroxide (Science Services) 1.5% potassium ferricyanide (Sigma)) for 30 min. After washes in CB and H₂O₂, cells were incubated in 0.5% aqueous uranylacetate (Science Services) over-night and dehydrated using a graded series of ice-cold ethanol-water composite. Cell monolayers were infiltrated in epon (Serva) and cured for 48 h at 60 °C. 50 nm ultrathin sections were generated on formvar-coated copper grids (Plano). Sections were imaged using a JEM-1400 + (JEOL) equipped with a XF416 (TVIPS) and the EM-Menu software (TVIPS) and analyzed using ShotMeister (JEOL).