background; were kindly provided by Dr. Marina Freudenberg (Max Planck Institute for Immunobiology, Freiburg and Professor Hans Hengartner (Institute for Experimental Immunology, University of Zu¨ rich). RNA preparations from the livers of C57BL/6 and IFNGR-deficient mice infected i.p. with Listeria monocytogenes strain EGD at 50% of the lethal dose were described earlier [4]. Experiments involving mice were performed following the guidelines given in Licence Nr. 50.203.2-K 13, 21/02 ‘‘Cell- biological basis of innate immunity’’ issued by the administrative authority of the City of Cologne. Serological reagents Primary serological reagents used were: anti-Irga6 165 rabbit antiserum (ref), anti-Irga6 mouse monoclonal antibodies (mAb) 10D7 and 10E7 (gifts from Jens Zerrahn, MPI for Infection Biology, Berlin), anti-calnexin rabbit antiserum (Biomol, Hamburg), anti- Gm130 mAb (BD Transduction Laboratory), anti-GRA7 5–241– 178 mouse mAb (gift from R. Ziemann, Abbott Laboratories). Secondary fluorescent antibodies were goat anti-mouse Alexa 546/ 488, goat anti-rabbit Alexa 546/488 (Molecular Probes). Northern blot analysis 5 mg of total RNA from liver or cells was separated on 1% agarose/formaldehyde gels and transferred onto Hybond-N+ membrane (Amersham Biosciences) by standard procedures. The blots were probed with randomly primed [a-32P]dCTP-labeled Irga6 ORF generated with the Rediprime DNA labeling system (Amer- sham Life Sciences). The similarly labeled ORF of mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control probe to reveal the amount of loaded total RNA, and the RNA Millenium Marker (Ambion) was used as a RNA size standard. Hybridizations were performed overnight at 42uC in a buffer containing 50% formamide, 56Denhardt’s solution (100 mg/ml each of Ficoll400, polvinylpyrrolidone and BSA Cohn Fraction V), 56standard saline-phosphate-EDTA (SSPE), 1% SDS, and 10% dextran sulfate. Membranes were washed under stringent conditions. The hybridization signal was detected by autoradiography, using Kodak X-OMAT AR films. Protein extraction and Western detection Mouse tissues or cells were snap frozen in liquid nitrogen, ground fine in a pestle and mortar (only for tissue), weighed and lysed in the following buffer (150 mM NaCl, 50 mM Tris, 1% NP- 40, 0.1% SDS, 0.5% deoxycholate, 5 mM EDTA, 1% Triton X- 100, pH 8.0) with protein inhibitor cocktail (Complete Mini, Roche) to a standard final concentration. The lysates were homogenized by repeated passage through a 20G syringe needle and cleared by centrifugation (30 min, 23,000 g). The superna- tants were then subjected to standard western blot analysis. Histology Tissue preparation. Mouse tissues were fixed in TBS/4% paraformaldehyde at 4uC and dehydrated through an ethanol series at 4uC (50%, 70%, 90%, 96%). Tissues were then transferred into isopropanol and finally into paraffin:isopropanol (1:1) solution at 60uC. The isopropanol was evaporated and fresh paraffin was then replenished several times at 60uC before the sections were moved to room temperature (RT). The embedded tissues were cut with a microtome RM 2065 (Leica Microsystems) into 6 mm thick serial sections. Immunohistochemistry. Paraffin sections were de-waxed in xylene and post-fixed in 4% paraformaldehyde for 1 h at RT. Expression of Irga6 in Liver Protein epitopes were then demasked by boiling for 10 min in 10 mM citrate buffer, pH 6.0. Non-specific protein binding and endogenous peroxidases were saturated with quenching buffer (1% BSA, 0.3% H2O2 in PBS) for 20 mins. After PBS washing, the sections were probed with 165/3 antiserum in DAKO diluent (DAKO, Hamburg). The HRP colour reaction was performed with the peroxidase substrate kit HistoGreen (Linaris). The nuclei were counterstained with Nuclear Fast Red. In situ hybridization. The Irga6 DNA template for the RNA probe were amplified using antisense (59-CGGGGAAGTCATAGT- GACAA-39) and sense primers (59-GGATCAGGGAAGTCCAG- CTT-39) and cloned into the vector pGEM-T-easy (Promega). The RNA-probes were synthesized using Sp6- or T7-RNA-polymerase and labelled with Digoxigenin (DIG) using the DIG labelling reaction mix (Roche). After de-waxing and post-fixation, paraffin embedded sections of mice liver were digested with 10 mg/ml Proteinase K in 0.1 M Tris pH 7.5 for 10 min at 37uC. The positively charged amino acids were blocked by 0.1 M triethanolamine pH 8.0, 0.25% acetic acid anhydride. Hybridization was performed overnight at 70uC in a buffer containing 50% formamide, 56SSC pH 7.0, 16Denhardt’s solution, 0.1% Tween20 and 0.1 mg/ml yeast tRNA (Roche). The slides were washed under stringent conditions. The hybridization was detected by probing with anti-DIG Fab fragments conjugated to alkaline phosphatase (Roche) using BM purple substrate (Roche) according to manufacturer’s instruction. Cell culture Established cell lines. L929 mouse fibroblasts, and TIB-75 hepatocytes were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM)supplemented with 10% FCS (Sigma-Aldrich, Deisenhofen, Germany), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were induced with mouse IFN-c (Cell Concepts) at a concentration of 200 U/ml for 24 h. Murine primary hepatocytes. Hepatocytes were isolated from liver using a two-step liver perfusion method (modified from [31]). Mice were anaesthetized with 2–5 ml Avertin and injected with 100 ml heparin-Na (5000 IU/ml). Mice were dissected and the liver portal vein was canulated. The liver was first perfused with pre-warmed (37uC) Krebs-Ringer-Buffer (KRB) (115 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 1.2 mM Na2SO4, 2.5 mM CaCl2, 25 mM NaHCO3, 10 mM glucose) at a flow rate of 3 ml/min for 30 min, then with pre- warmed 50 ml KRB with 1 mM EDTA, and finally with 100 ml KRB with 0.5 mM CaCl2 and 0.05% collagenase (SIGMA) at the same flow rate. Hepatocytes were liberated from the liver capsule and filtered through 100 mm gauze. The cell suspension was washed twice at 4uC with washing buffer (1xHanks buffer, 0.5% BSA, 10 mM HEPES pH 7.65) at low speed (50 g) and then twice with DMEM/Ham’s F12 1:1 Medium (Supplemented with 10% FCS, insulin, transferrin and selenium (ITS, Roche) at 1%, 0.1 mM dexamethasone and 100 U/ml Penicillin/100 mg/ml streptomycin buffered by 15 mM HEPES). Cells were then pre- seeded for 15 min on collagen coated plates to eliminate tightly adherent cells and then transferred to other collagen coated plates for culture for several days. Note: All buffers and gauze were sterilized and great care was taken when handing the cells to avoid high mortality. Immunofluorescence procedures Immunofluorescence of cultured cells was performed as described previously [32] and examined with an Axioplan II fluorescence microscope equipped with an AxioCam MRm camera using the Axiovision 4.7 software (all Zeiss). PLoS ONE | www.plosone.org 11 August 2009 | Volume 4 | Issue 8 | e6787