A horseradish peroxidase (HRP)-PF-02341066 ic50 conjugated goat anti-rabbit IgG (Nichirei Biosciences, Tokyo, Japan) was used as the secondary antibody. Peroxidase visualization was done using 3,3′-Diaminobenzidine (DAB). All techniques including H&E staining were performed by Animal Pathology Platform, Biomedical Research Core of Tohoku University Graduate School of Medicine. Cell sorting and phenotyping of murine stromal cells TFK-1 xenografts were

used in this experiment. Freshly isolated subcutaneous tumors of NOG-EGFP mice were dissociated by mincing the tissue with scalpels, followed by incubation in RPMI-1640 media containing collagenase (Worthington Biochemical, NJ, USA) for 30 min at 37°C. After incubation, the cell suspension was filtered through selleck inhibitor a 100-μm cell strainer. The cells were resuspended in phosphate buffered saline (PBS) and sorted on a fluorescence-activated selleck screening library cell sorter (FACS Aria TM II Cell Sorter, BD Biosciences, Erembodegem, Belgium) on the basis of single-cell viability and the presence of GFP. For immunophenotyping, cells were incubated for 30 min at room temperature with conjugated antibodies against mouse CD31, CD90, CD49b, CD14, CD11c (CD31: 561410, CD90: 553007, CD49b: 553858, CD14: 560636 and CD11c: 560583, BD Biosciences) or conjugated isotype controls (APC-CyTM7 (Rat IgG1, κ)-560534,

Alexa-Flour700 (Hamster IgG, λ1): 560555, APC (Rat IgG2a, κ): 53932, PE (Rat IgM, κ): 553943, PE-CyTM7 (Rat IgG2a, κ): 552867, BD Biosciences), as previously reported [6] . Analyses were performed on a FACS Aria TM II Cell Sorter (BD Biosciences). Viability of sorted cancer cells Xenografted tumors of TFK-1 cells in NOG-EGFP mice were harvested and separated into cancer cells and stromal cells by FACS as described above. Collected TFK-1 cells were cultured on dishes and subsequently reimplanted in NOG-EGFP mice. Ribonucleotide reductase In order to confirm the effect of removal of eGFP-expressing cells, the subcutaneous tumors of TFK-1 cells were provided for primary cell culture without FACS sorting as a control. Statistical analysis Data were presented as the mean ± S.E. Statistical significance was determined by Mann–Whitney U test performing using GraphPad Prism for Windows version 5.02.

Differences between experimental groups were considered significant when the p-value was <0.05. Results Confirmation of eGFP expression in NOG-EGFP mice Green fluorescence was detected in the NOG-EGFP mice by a hand-held UV lamp (Figure 1A). Almost all internal organs showed green fluorescence in the imaging instrument (Figure 1B). The fluorescence of skin fibroblasts was visible using a fluorescence microscope (Figure 1C). Histological findings revealed eGFP-expressing cells (shown as DAB-positive cells in Figure 1Db and fluorescent cells in Figure 1Dc) in the stroma of the xenografted tumors, whereas cancer cells did not show eGFP expression (Figure 1Db-c). Based on the findings mentioned above, expression of eGFP on NOG-EGFP mice was confirmed.