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Transmission electron microscopy Hepatic stem like cells and mature hepatocyte like cells derived from SSCs were fixed in 2. 5% wv glutaraldehyde in 0. 1 M cacodylate buffer. After extensive washing in PBS, the cells ABT-888 ic50 were post fixed in 1% wv OsO4 for 30 min, dehydrated in a graded solution of ethanol and embedded in Epon. Ultrathin sections were cut and examined under an electron microscope after staining with uranyl acetate and lead citrate. Immunocytochemistry Immunocytochemistry was performed on SSCs, the transdifferentiated cells, and the fully differentiated cells according to the procedure described previously. The cells were fixed with 4% paraformaldehyde and perme abilized in 0. 4% triton X 100 for 15 min.<br><br> After washing with phosphate buffered saline, cells were Afatinib 分子量 blocked in 1% bovine serum albumin for 15 min and followed by incubation with primary antibodies at a dilution with 1 100 overnight at 4 C. Primary antibodies used were anti VASA, anti RET, anti UCHL1, anti GFRA1, anti OV6, anti CYP1A2, anti ALB, anti PLZF, anti SSEA 1, anti SSEA 4, anti Nanog, or anti TRA 1 81. After three washes with PBS, the cells were incubated with the secondary antibody, including FITC conjugated or rhodamine conjugated IgG, at a 1 200 dilution for 45 min at room temperature. DAPI was used to stain the nuclei, and the cells were observed for epifluorescence using fluorescence microscope. Double staining was performed to determine whether the cells derived from SSCs were co expressing CK19 and CK8 using anti CK19 and anti CK8.<br><br> Immunofluorescence was also carried out to deter mine the expression changes of phosphorylation of ERK12, Smad2, Stat3, and Akt in the C18 4 cells, the transdifferentiated cells, and differentiated cells using anti bodies against phospho ERK12, phospho Smad2, phospho Stat3, or phospho Akt. RNA extraction and reverse transcription polymerase chain reaction AG-1478 価格 Total RNA was extracted from C18 4 cells, the trans differentiated cells, and the fully differentiated cells from C18 4 cells and primary SSCs using Trizol. Reverse transcription was performed using First Strand cDNA Synthesis Kit and PCR was performed according to the protocol as described previously. The forward and reverse primers and PCR products of the chosen genes, including Cytokine 8, Ck18, Ck7, Ck19, Cyp1a2, Cyp7a1, hepatocyte nuclear factor 3b, Hnf4a, Albumin, tyrosine aminotransferase, transthyretin, Cyclin A, Cyclin B, Cyclin D1, Cyclin E, CDK1, CDK2, c fos, Oct 4, and Gapdh were designed and listed in Additional file 1Table S1.<br><br> The PCR reaction started at 94 C for 5 min and was performed as followsdenaturation at 94 C for 30 sec, annealing at a temperature as indicated in Additional file 1Table S1 for 45 sec, and elongation at 72 C for 45 sec. After 30 cycles, the samples were incubated for an additional 5 min at 72 C. PCR products were separated by electrophoresis on 1. 2% agarose gels. The gels were exposed to chemilu minescence. Flow cytometry Hepatic stem like cells derived from SSCs in the condi tioned medium with or without MEK1 inhibitor PD98059 were first fixed with 1% paraformaldehyde, permeabilized by permeabilization buffer, and incubated with primary antibody to CK8.