Cm3050s kryostat

To fixate tdTomato in the tissue, harvested organs were incubated in ROTIHistofix 4% for 24h at room temperature (RT), then transferred to 30% sucrose solution and incubated 24h at 4°C. Tissue was embedded and sectioned in cryo‐mounting medium (Sakura OCT) at Leica CM3050S Kryostat. Immunofluorescence staining was performed using rabbit anti‐Lysozyme (Dako, Catalogue A0099, 1:2000), rabbit anti‐OmpC (biorbyt, orb6940, 1:1000) with goat anti‐rabbit‐biotinylated (Dianova, Catalogue 111‐065‐144) with Streptavidin Protein‐DyLight 550 system and mouse anti‐NeuN (Millipore, Catalogue MAB377, 1:300) with Alexa Fluor 647 goat anti‐mouse (Invitrogen, Catalogue A21235). In some experiments Paneth and goblet cells were stained with Ulex europaeus agglutinin I (UEA‐1) (Vector laboratories, Cat. No.: FL‐1061). Nuclei counterstaining on tissue‐sections was performed using Hoechst 33342 (Invitrogen) or DAPI (Sigma‐Aldrich). Images were obtained using a confocal fluorescence microscope (LEICA TCS SP5 II) together with the LEICA DFC360 FX camera and the imaging software ‘LAS X’ (Leica) or a confocal inverted microscope (Nikon Eclipse TE2000‐E) and NIS‐elements imaging software (version 4.13.05) and ImageJ (https://imagej.nih.gov/ij/).

Seven-week old C57BL/6J mice (Jackson Laboratories) were infected by retro orbital i.v. injection with ~500 CFU of Salmonella Typhimurium SL1344 SopB^3xFLAG or SipA^3xFLAG (4 mice per strain). Mice were monitored daily for signs of clinical illness. On days 4 and 5, animals were anesthetized with isofluorane prior to transcardial perfusion with pharmaceutical grade heparin/saline (100 U/mL), followed by perfusion with 4% (w/v) PFA. Gallbladders were harvested and post-fixed in 4% (w/v) PFA for 4 h at RT, washed three times with PBS (30 min each) and cryopreserved overnight in 30% (w/v) sucrose/PBS at 4°C. The following day, gallbladders were incubated in ‘optimal cutting temperature’ compound (OCT; Sakura Finetek) for 15–30 min (RT) and 5 μm sections were prepared on Shanodon Positively Charged Superfrost slides (Thermo Scientific) using a Leica CM3050S Kryostat. Slides were air-dried and stored at -20°C until staining.