Cells for intracellular staining were pretreated with brefeldin A (10 μg/mL) and permeabilized. Stained cells were analyzed using FACS Canto II (Becton Dickinson, NJ), and the data were analyzed using FlowJo software (Tree

Star, Ashland, OR). Liver specimens were fixed with 10% formalin, paraffinized, and sectioned to 6 μm thickness, then deparaffinized and rehydrated. Antigen retrieval was obtained by boiling in 10 nM citrate buffer solution. After blocking with 20% goat serum, sections were embedded with primary antibodies against CCR9 (Abcam, ab1662, Cambridge, UK) selleck chemicals llc and alpha smooth-muscle actin (α-SMA) (DakoCytomation, clone 1A4, Carpinteria, CA) or CCR9 and F4/80 (eBioscience, clone BM8, San Diego, CA)

overnight. Sections were then stained with secondary antibodies labeled with Alexa Fluor 488 or Alexa Fluor 568 (Life Technologies, Carlsbad, CA), and nuclei counterstained with DAPI (Vector Laboratories, Burlingame, CA). Immunohistochemistry was performed with mAb to α-SMA (DakoCytomation) using an M.O.M. kit (Vector Laboratories).9 Total RNA was extracted from liver homogenates or cultured cells using Trizol reagent (Gibco-BRL, Grand Island, NY). Complementary DNA was synthesized from 100 ng of total RNA by reverse transcription. click here PCRs were performed using AmpliTaq Gold Fast PCR MasterMix (Applied Biosystems, Foster City, CA) and the predesigned primers listed in Supporting Table 2. To quantify the products, real-time PCR was performed using TaqMan Universal Master Mix

and StepOne Plus systems (Applied Biosystems). The level of target gene expression was normalized to β-actin expression in each sample. LSECs were isolated as previously described.28 Details are described in the Supporting Methods. Isolation of HSCs and information regarding coculture or treatment are described in the Supporting Methods. The cells were cocultured for 24 hours and HSCs were harvested from a plate using 0.25% EDTA trypsin, after macrophages or other cocultured cells were washed away. Purity over 97% of HSCs was confirmed by flow cytometry. HSC RNA was isolated for qPCR as described above. Cell-migration assays were performed using 8 μm-pore 96-well Transwell plates (Corning, Corning, NY). Serum-starved HSCs PI-1840 or isolated hepatic CD11b+ cells from WT or CCR9−/− mice were placed in the upper chamber and exposed to CCL25 (R&D Systems, no. O35903.1) at the indicated concentrations in the lower chamber. After 48 hours of incubation at 37°C, cells that migrated to the lower chamber were counted. Data were analyzed using JMP9 (SAS Institute, Cary, NC) and expressed as mean ± standard error of the mean (SEM). The Mann-Whitney U test, the unpaired Student t test, and the Kruskal-Wallis test were used to assess the differences between groups, as appropriate. Differences were considered statistically significant when P < 0.05.