4G2) to prevent nonspecific binding, five-color flow cytometry was conducted via the 30-minute incubation of the cells with fluorochrome-conjugated monoclonal antibodies. Intragraft T cell apoptosis was detected with an annexin V–PE apoptosis detection kit (BD Pharmingen) according to the manufacturer’s protocol. Flow analysis was performed with an LSR II flow cytometer (BD Biosciences). The analysis of human tissue was carried out according to the

University of Pittsburgh institutional review board protocol (PRO10110393). Formalin-fixed, paraffin-embedded human liver allograft find more biopsy sections were obtained from 3 normal livers and 16 postreperfusion biopsy samples (1-4 hours), and they were analyzed with multiplex QD–based immunofluorescent staining for the evaluation of B7-H1 expression on specific cell types.21 Briefly, 4-μm sections were deparaffinized, hydrated, and treated with citrated buffer antigen retrieval. Triplex or

quadruplex staining was performed with sequential incubation cycles of blocking, primary antibody incubation, biotinylated secondary antibody incubation, Dabrafenib clinical trial and streptavidin-coated QD incubation. For each cycle, sections were blocked with an avidin and biotin block kit (Vector Laboratories, Inc., Burlingame, CA) and a protein block (Dako, Carpinteria, CA). Primary antibodies included rabbit anti–B7-H1 (LifeSpan Bioscience, Seattle, WA) and anti-CD11c (Abcam, Cambridge, MA), mouse anti-CD31, anti-CD68, anti-HepPar1 (Dako), and anti-cytokeratin 19 (CK19; Santa Cruz Biotechnology, Santa Cruz, CA). After all antibodies were stained and Hoechst nuclear staining selleck chemical was applied, digital images of whole stained slides were obtained with MIRAX MIDI digital whole slide scanning systems (Carl Zeiss MicroImaging, Jena, Germany) and were analyzed with Pannoramic Viewer (3D Histech, Ltd., Ramsey, NJ). Human hepatocytes were isolated from histologically

normal livers with a three-step collagenase perfusion technique (Dr. Steven Strom and Dr. Ken Dorko, University of Pittsburgh Core Pathology Facility, Pittsburgh, PA) according to an institutional review board–approved protocol. After an overnight culture, hepatocytes in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum were exposed to hypoxia (1% O2) at 37°C and were harvested after 1 to 6 hours for RNA isolation and RT-PCR with primers for human B7-H1 (forward, 5′-CTGTCCGCCTGCA GGGCATT-3′; reverse, 5′-AACAGCCGGGCCCTCT GTCT-3′). The data are presented as means and standard errors of the mean. Comparisons between the groups at different time points were performed with the Student t test or an analysis of variance with StatView (Abacus Concepts, Inc., Berkeley, CA). Differences were considered significant at P < 0.05. The modulation of B7-H1 expression on both hepatocytes and NPCs has been shown after inflammatory stimulation.