5 system (Roche Diagnostics). The reaction was performed in 2 μL cDNA for each analyzed sample using selleck chemicals the LightCycler FastStart DNA Master HybProbe Kit (Roche Diagnostics).

Primers and Probes were S1P1 (sense: 5′-GTTTCTGCGGGAAGGAAGTA-3′, antisense: 5′-AGCA AGGAGGCTGAAGACTG-3′ and Universal Probe Library [UPL] probe no. 21; Roche), S1P2 (sense: 5′-CCTGGT CACCGACTCCTG-3′, antisense: 5′-GGCATATGCAAG CCTCTCTC-3′, and UPL probe no. 78), and S1P3 (sense: 5′-ACTTAGCGGTGGCAGCAT-3′, antisense: 5′-GAAAC AGGCTCTCGTTCTGC-3′, and UPL probe no. 26). Real-time PCR for rat Rho, rat Rho kinase, mouse S1P receptors, and mouse smooth-muscle α-actin was performed using the 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA) and according to the TaqMan method in a 25 μL volume containing 12.5 μL 2 × TaqMan Universal Master Mix, No AmpErase UNG (Applied Biosystems) and 2 μL cDNA. Primers and probes of rat Rho, Rho kinase, and mouse S1P receptors were S1P1 (sense: 5′-TTTA GCCGCAGCAAATCAGA-3′, antisense: 5′-GGTTGT CCCCATCGTCCTT-3′, probe:

5′-AACTCCTCTCA CCCCC-3′), and others as described.17, 18 Mouse smooth-muscle α-actin primers and probe were obtained from Applied Biosystems, TaqMan Gene Expression Assays (Mm00725412_s1). Each target gene expression was normalized with endogenous control gene. Hepatic stellate cells were isolated from rats weighing 300 to 400 g as described,19 with some modification using Optiprep (Axis-Shield PoC AS, Oslo, Norway),20 and cultured on uncoated plastic tissue-culture dishes (Falcon, Lincoln Park, NJ). Excised liver specimens MCE buy PD98059 were fixed immediately in 10% formalin and embedded in paraffin, or were snap-frozen in OCT compound. Serial 4-μm-thick liver tissue sections were deparaffinized and analyzed by hematoxylin-eosin and Sirius Red staining for collagen. Cryosections were fixed and first stained using the β-Galactosidase Staining Kit (Mirus Bio, Madison, WI).11 Then Sirius Red staining or immunohistochemical analysis

for smooth-muscle α-actin was performed using a Vector M.O.M. Immunodetection Kit (Vector Laboratories, Burlingame, CA) in accordance with the protocol specified by the manufacturer, with a ready-to-use mouse monoclonal antibody (PROGEN Biotechnik, Heidelberg, Germany). Sections were counterstained with nuclear fast red. Serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and gamma-glutamyltransferase were determined using an automated analyzer (Bio Majesty JCA-BM 8040, JEOL, Tokyo, Japan). Quantitative data are presented as means ± standard error of the mean (SEM). Comparisons between groups were made using Student t test. Statistical significance was set at P < 0.05. The hemodynamic effects of S1P2 antagonist were examined in rats with bile duct ligation and with sham operation at 4 weeks after the operation.