10 week old mice of mixed genetic background (DBA/C57Bl/6) and GFAP-Cre mice were used as controls. All

mice received a single i.p. injection of BrdU (10 mM, 1 ml per 100 g bodyweight) 2 h before killing. Histology and immunohistochemistry Liver samples were either quick-frozen in liquid nitrogen and stored at -80°C or fixed in 4% paraformaldehyde and routinely embedded in paraffin. Frozen liver samples were used for PECAM1 immunohistochemistry and were processed as described [16]. For all other antibodies (Table 1) and hematoxylin-eosin DAPT in vitro (HE) staining 2 μm paraffin sections were used and processed as described [16] Antigen-antibody complexes were detected by peroxidase- or Cy-2/3-conjugated secondary antibodies as previously described [41, 42]. Similarly processed liver slides where the primary antibody was omitted

were used as negative controls. Monoclonal mouse antibodies were used together with the Vector M.O.M. Immunodetection Kit (Vector Laboratories, CA, USA) to avoid a cross-reactivity of secondary antibodies with endogeneous immunoglobulins of mouse tissue. For detection of Kupffer cells (the liver specific macrophages), the anti-F4/80 antibody was used instead of an antibody against the macrophage/monocyte marker CD14. Isolation of liver cells and cell culture Hepatocytes were isolated using an in vitro perfusion technique [43]. Liver was perfused with calcium free buffered saline and subsequently with collagenase (1 mg/ml, 240 U/mg, Biochrom AG, Berlin, Germany). Cell suspension was this website centrifuged thrice Phospholipase D1 at 70 × g, 5 min. Sinusoidal cells were isolated by perfusing liver consecutively with calcium free buffered saline, pronase (1 mg/ml) and collagenase (1 mg/ml) for 10 min each. Cell suspension was centrifuged twice at 70 × g disposing the hepatocytes and twice at 250 × g for washing and collecting sinusoidal cells. Cells were re-suspended and either undergone RNA isolation or incubated with anti-CD146 antibody linked to magnetic beads according to the suppliers recommendation

(Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). CD146 positive SECs were eluted after magnetic separation. After two washings RNA was extracted. Isolation of RNA and quantitative real time RT-PCR (Q-RT-PCR) Total RNA was isolated using the PeqGOLD RNA Pure isolation system (Peqlab, Erlangen, Germany). Quality of RNA was assessed by electrophoresis in denaturing formaldehyde agarose gels and purity was estimated by ratio A260/280 nm spectrophotometrically. Concentration was adjusted to 0.5 mg/ml. RT-PCR for real time quantification was performed as previously described [42] using primers listed in Table 2. RNA sample load was normalized using amplifications with the housekeeping gene cyclophilin. Standard curves of serial dilutions from total RNA were used for transforming the ct-values in concentration values depicted as arbitrary units. For primer design of total M-Pk and M2-Pk the RNA sequence [Genbank: NM_011099] was used.