Chen et al. Journal of Experimental & Clinical Cancer Research 2010, 29:13 http://www.jeccr.com/content/29/1/13 Page 3 of 8 respectively injected into the tumors for every 3 day. The delivery efficiency was evaluated by GFP fluorescence and RT-PCR. After 3 weeks the mice were killed (with permis- sion of the Animal Protection Association of Tongji Medi- cal College). Tumors were cryosectioned or fixed in 10% buffered formalin and embedded in paraffin detected by immunohistochemistry. Western blot analysis IGFBP7 expression changes within mouse xenografts were checked by western blotting as described pre- viously [19], the antibodies to IGFBP7 and b-actin were purchased from (R&D systems U.S.A.). Detection of IGFBP7, caspase-3, VEGF by immunohistochemistry or laser scanning confocal microscopy Detection is based on the formation of the Avidin-Biotin Complex (ABC) with primary antibodies that reacted with tissue antigens. Primary antibodies were listed as follows: IGFBP7(1:25 R&D systems U.S.A MAB21201), caspase-3(1:20 R&D systems U.S.A MAB835), VEGF (1:20 Santa Cruz Biotechnology, sc-7269). Coverslips containing pcDNA3.1-IGFBP7, pcDNA3.1-CONTROL tumor section were mounted onto glass slides and observed with a Zeiss 510 confocal microscope. Green fluorescent protein and TRITC-labeled IGFBP7 were viewed through the GFP, and tetramethyl rhodamine isothiocyanate (TRITC) fluorescence channel, respec- tively. Appropriate positive and negative controls were included. The expression of caspase-3 and VEGF visuali- zation is based on enzymatic conversion of a chromo- genic substrate (AEC), (CTS018 R&D systems U.S.A). No significant difference in intensity of immunohisto- chemical staining was designated as negative (0), positive (1), strong positive (2) and the percentage of positive cells was scored as less than 5% (0), 5%~25% (1), 26% ~50% (2), 51%~75% (3) or over 75% (4) of cells stained [20]. Values in the parentheses were multiplied together to the scores for IGFBP7, caspase-3, VEGF expression. Detection of tumor apoptosis Tumor apoptosis was detected using terminal deoxynu- cleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL, Catalog # 11684809910, ROCHE Germany) according to the supplier’s instruc- tions, and apoptosis index (AI) was used to evaluate cell apoptosis. Statistics The statistical analysis was performed using SPSS 13.0 software (SPSS, Chicago, IL, U.S.A.). Statistical compari- sons of mean values were performed using Student’s t-test and Kruskal-Wallis Test, the correlations was analyzed by Spearman’s rho correlation analysis. All P-values were determined from two-sided tests. A signif- icance criterion of P < 0.05 was used in these studies. Results Identification of pcDNA3.1-IGFBP7 plasmid The sequence analysis of constructed pcDNA3.1- IGFBP7 by a DNA sequencer showed the same sequence of eukaryotic IGFBP7 mRNA as designed. Meanwhile, recombinant pcDNA3.1-IGFBP7 plasmid was confirmed by restriction enzyme analysis, as shown in additional files 1, Figure S1. These results indicated that the pcDNA3.1-IGFBP7 vector was constructed suc- cessfully. Then pcDNA3.1-IGFBP7 and pcDNA3.1- CONTROL were transfected into cells successfully, termed pcDNA3.1-IGFBP7 cells and pcDNA3.1-CON- TROL cells, respectively with transfection rate being about 60%, as shown in additional files 1, Figure S2. Effect of pcDNA3.1-IGFBP7 plasmid on IGFBP7 expression It was found that the IGFBP7 mRNA levels in pcDNA3.1- IGFBP7-transfected B16-F10 cells were increased by about 4-fold, 8-fold, 7-fold, 6-fold on days 1, 3, 6 and 12, respec- tively, compared with the control group. But no change of IGFBP7 expression in pcDNA3.1-CONTROL groups (P > 0.05) was found, suggesting that pcDNA3.1-IGFBP7 vector specifically promotes expression of IGFBP7 without effects on b-actin mRNA,, as shown in additional files 2, Figure S1. Meanwhile, the expression of IGFBP7 was detected by western blot. The western blot showed that pcDNA3.1-IGFBP7 increased the expression of IGFBP7. Results are consistent with previous determined by RT-PCR. According to these results detected by RT-PCR and western blot, the IGFBP7 expressed in the pcDNA3.1- IGFBP7 group were significantly higher in the pcDNA3.1- CONTROL and B16-F10 cells groups (p < 0.03), as shown in additional files 2, Figure S2. pcDNA3.1-IGFBP7 suppresses B16-F10 cells growth in vitro The proliferation of pcDNA3.1-IGFBP7-transfected cells was significantly suppressed compared with control cells (P < 0.01). The highest suppression effect of pcDNA3.1- IGFBP7 was found at 48 h post-transfection, and no signif- icant difference in proliferation between pcDNA3.1-CON- TROL and untransfected cells was observed (P > 0.05), indicating that transfection of pcDNA3.1-IGFBP7 blocks the proliferation of B16-F10 cells by increasing IGFBP7 synthesis and secretion, as shown in additional files 2, Figure S3. To evaluate apoptosis-induced effect of pcDNA3.1-IGFBP7 in melanoma cells, B16-F10 cells at 48 h post-transfection was monitored by FCM. The apoptosis rate in pcDNA3.1-IGFBP7 group (24.6%) was significantly higher than that in control groups (P < 0.01). However, no