Primer sequences were as follows: DKK-1, 5′-TCACGCTATGTGCTGCCCCG-3′ and 5′-TGAGGCACAGTCTGATGACCGGA-3′, product size 223 bp; and GAPDH, 5′-AGAAGGCTGGGGCTCATTTG-3′ and 5′-AGGGGCCATCCACAGTCTTC-3′, product size 258 bp. PCRs were optimized for the number of cycles to ensure product intensity to be within the linear phase of amplification. The PCR protocol consisted of an initial denaturation step

of 95°C for 7 minutes, followed by 32 cycles of a three-step program of 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 45 seconds, and a final extension step of 72°C for 7 minutes. The PCR was performed in a final volume of 25 μl in the presence of 2.0 mM MgCl2, 0.75 U of Taq polymerase in PCR buffer, and 5 buy KPT-8602 pmol of the hDKK-1 and GAPDH primers. PCR products were separated and analyzed on 1.5% agarose gels. Elisa Levels of DKK-1 in cell medium, cell lysate, serum, and cerebral fluid were measured by ELISA with a commercially available enzyme test kit (R&D Systems,

Inc.) according to the supplier’s recommendations. First, a rabbit polyclonal antibody specific to DKK-1 was added to a 96-well microplate as a capture antibody and incubated overnight at room temperature. After washing away any unbound antibody, 0.75% BSA was added to the wells and incubated for at least INK1197 cell line 1 h at room Selleck A1155463 temperature for blocking. After a wash, 3-fold diluted sera were added to the wells and incubated for 2 h at room temperature. After washing away any unbound substances, a biotinylated polyclonal antibody specific for DKK-1 was added to the wells as a detection antibody and incubated for 2 h at room temperature. After a wash to remove any unbound antibody-enzyme reagent, horseradish peroxidase (HRP)-streptavidin was added to the wells and incubated for 20 min. After a wash, a substrate solution was added to the wells and Glutathione peroxidase allowed to react for 20 min. The reaction was stopped by adding 50

μL of 2 N sulfuric acid. Color intensity was determined by a photometer at a wavelength of 490 nm, with a reference wavelength of 570 nm. Differences in the levels of DKK-1 between different groups were analyzed by t test. Significance was defined as P < 0.05. Immunohistochemistry To investigate the DKK-1 protein in clinical samples that had been embedded in paraffin blocks, we stained the sections as previously described [17]. Briefly, 3.3 μg/mL of a rabbit polyclonal anti-hDKK-1 antibody (Santa Cruz Biotechnology) were added to each slide after blocking of endogenous peroxidase and proteins, and the sections were incubated with biotin-labeled anti-rabbit IgG as the secondary antibody. Substrate-diaminobezidine (DAB) was added, and the specimens were counterstained with hematoxylin. Statistical analysis Statistical analyses were done using the SAS6.12 statistical program. Kendall’s tau-c association analysis was applied between DKK-1 expression and pathologic tumor classification.