, 1991), which harbors a site-specific Tn7 transposase, were used for conjugational transfer to Yersinia. All constructs were verified by PCR and DNA sequencing. Yersinia and E. coli were routinely PD-166866 ic50 grown in Luria–Bertani broth (LB) at 27 and 37 °C, respectively. Chloramphenicol (20 μg mL−1), nalidixic acid (60 μg mL−1), and kanamycin (50 μg mL−1) were used as selective antibiotics. Escherichia coli DH-5α (Hanahan, 1983) was used as the primary host in cloning experiments; E. coli S17.1 λpir (Simon et al., 1988) was used as a donor for conjugation. Bioluminescent yersiniae were grown in LB medium at 27 °C with shaking to the late exponential phase, washed twice, and

resuspended in an LB medium containing 15% of glycerol. Bacteria were stored at −80 °C and the CFU were determined by plating serial

dilutions. 6–8-week-old female BALB/c mice were orally infected with 1 × 109 CFU Yersinia using a microliter selleck compound pipette or intravenously into the lateral tail vein with 1 × 104 CFU. Infection was followed daily for up to 6 days using the IVIS Lumina System (Xenogen). To induce luminescence of yersiniae, mice were intraperitoneally injected with 120 mg l-arabinose in phosphate-buffered saline as described previously (Loessner et al., 2007). Before imaging, mice were anesthetized with isoflurane using the Xenogen Gas Anesthesia System XGI-8. After live imaging, mice were sacrificed by CO2 asphyxiation and the entire intestinal tract was removed along with the liver, spleen, mesenteric, and cervical lymph nodes and subjected to analysis using the IVIS Lumina system. Statistical significance of the data was determined using a two-tailed Mann–Whitney test. P≤0.05 was considered significant. Culturing yersiniae from different organs revealed 99% stability of the luciferase construct for at least

5 days in the mouse model. Small intestines with PPs, cervical lymph nodes, and spleen were embedded in Tissue-Tek (Sakura Finetek) and shock frozen in liquid nitrogen. Cryosections of 10 μm thickness were prepared using a Leica Cryomicrotome for CM3050 and mounted on SuperFrostPlus slides. Cryosections were immunostained as described previously (Halle et al., 2007; Oellerich et al., 2007). Yersiniae were stained by a primary polyclonal rabbit antibody, followed by a goat anti-rabbit Alexa Fluor 555 (Invitrogen)-coupled antibody (red). T-cells were stained with a hamster anti-CD3e primary antibody, followed by a goat anti-hamster Cy2 antibody (green). B-cells were stained by a rat anti-B220 primary antibody, followed by a goat anti-rat Alexa Fluor 647 (Invitrogen)-coupled antibody (pink). Granulocytes and polymorphonuclear leukocytes were stained with a rat anti-mouse Ly6C/G antibody, followed by goat anti-rat Alexa Fluor 647 (Invitrogen) anti-rat antibody (pink). Primary antibodies were obtained from Beckton Dickinson.