The PCR conditions were as follows: incubation at 94°C for 4 min; 35 cycles of incubation at 94°C for 50 s, 60°C for 30 s, and 72°C for 1 min; with a final incubation at 72°C for 10 min. PCR products were separated using 2% agarose gel electrophoresis and stained with ethidium bromide. Labeling stem cells with PKH26 PKH26 is a red fluorochrome. It has excitation (551 nm) and emission (567 nm) characteristics compatible with rhodamine or phycoerythrin detection systems. The linkers are physiologically stable and show little to no toxic side-effects on cell systems. Labeled cells retain both biological and proliferating
activity, and are ideal for in vitro cell labeling, in vitro proliferation studies and long term, in vivo cell https://www.selleckchem.com/products/gm6001.html tracking. In the current work, undifferentiated MSCs cells were labeled with PKH26 according to the manufacturer’s recommendations (Sigma, Saint Louis, Missouri,
USA). Cells were injected intravenously into rat tail vein. After one month, liver tissue was examined with a fluorescence microscope to detect the cells stained with PKH26. Fluorescence was only detected in the 5th rat group. Real-time quantitative analyses for β-catenin,PCNA,cyclin D and survivin genes expression Total RNA was extracted from liver tissue homogenate https://www.selleckchem.com/products/Temsirolimus.html using RNeasy purification reagent (Qiagen, Valencia, CA). cDNA was generated from 5 μg of total RNA extracted with 1 μl (20 pmol) antisense primer and 0.8 μl superscript AMV reverse transcriptase for 60 min at 37°C. Quantitation of gene
expression was conducted using universal probe library sets based real time PCR (Roche diagnostics). Selection of genes specific probes and primers were done PAK6 using the online ProbeFinder software and the real time PCR design assay of Roche Diagnostics found their website: http://www.universalprobelibrary.com, Hypoxanthine phosphoribosy-ltransferase 1 (Hprt1) was used as a positive control house keeping gene. FastStart Universal Probe Master mix was used in LightCycler® 480 Instrument (Roche Applied Science, Indianapolis, USA). Briefly, in the LightCycler® 480, a total reaction volume of 20 μl was prepared, of which 2 μl of starting RNA material was included for RT-PCR, a final concentration of 0.5 μM of each forward and reverse primer and 0.2 μM of the TaqMan probe was used. Cycling conditions involve reverse transcription at 50°C for 30 min; enzyme find more activation at 95°C for 15 min, followed by 50 cycles of 95°C for 10 sec and 60°C for 60 sec. LightCycler® 480 RT-PCR data were analyzed using LightCycler1.2 version 3.5 software using the second derivative maximum method. Successfully amplified targets are expressed in Ct values, or the cycle at which the target amplicon is initially detected above background fluorescence levels as determined by the instrument software.