For each tumor section, quantification of immunofluorescence double staining was performed by counting Ki-67+ cells in six high power fields (400 × magnification) in parallel with LgR5+. The proportion of Ki-67 positivity in counted LgR5+ cells was expressed in percentages. Real-time quantitative reverse transcription-PCR analysis To analyze gene expression of LgR5 by RT-PCR, we extracted total cellular RNA and performed cDNA Torin 2 in vitro synthesis using the Absolutely RNA FFPE

Kit and AffinityScript QPCR cDNA Synthesis Kit from Stratagene (Waldbronn, Germany). Areas of interest (only epithelial regions) for each tissue section were manually microdissected using a scalpel blade. For both groups (BE and EAC

without BE) equal amounts of tissue areas were assessed (2 × 1.5 cm2 surface area per section, thickness of 10 μm). RNA extraction and cDNA synthesis ISRIB chemical structure were performed according to the manufacturer’s instructions. For OE-33 cell line, after homogenization Diethyl pyrocarbonate (DEPC)-75% ethanol was added to the lysate to provide ideal binding conditions. Primers were designed using the Primer Express software for primer design to amplify short segments of 50-150 base pairs of target cDNA. The LgR5 forward primer sequence was: 5′-TGCTGGCTGGTGTGGATGCG-3′; the LgR5 reverse primer sequence was: 5′-GCCAGCAGGGCACAGAGCAA-3′. Matched human esophageal cDNA was purchased by BioChain (Hayward, CA, USA) as control. The housekeeping gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) selleck inhibitor was used for relative quantification and cDNA quality control. The GAPDH forward primer sequence was: 5′-ATCCCATCACCATCTTCCAGG-3′; the GAPDH reverse primer sequence was: 5′-CGCCCCACTTGATTTTGG-3′. All PCR reactions were carried out with a DNA Engine Opticon 2 System

(MJ Research, old Biozym, Oldendorf, Germany). Total RNA was reversely transcribed into cDNA according to the manufacturer’s manual. Each PCR reaction was performed in 25 μl volume containing 12.5 μl the Sensi Mix (Peqlab, Erlangen, Germany), 0.5 μl SYBR Green, 10 pmol/μl forward primer, 10 pmol/μl reverse primer, 1 μl template DNA (150 ng) and 9 μl peqgold RNAse free water. Initial denaturation at 95°C for 10 minutes was followed by 38 cycles of a denaturation step at 95°C for 15 seconds, an annealing step at 60.9 °C for 30 seconds, and an extension step at 72°C for 40 seconds. To confirm amplification specificity, the PCR products from each primer pair were subjected to a melting curve analysis. Negative controls without template were produced for each run. Quantification data were analyzed using the LightCycler analysis software. Reproducibility was confirmed by independent PCR repeated twice. The average threshold cycle (Ct) value was calculated as the cycle number at which the fluorescence of the reporter reaches a fixed threshold.