Alexa fluor 647 monoclonal antibody labelling kit

To examine the localisation of infiltrated GFP-positive bone marrow-derived cells and F4/80 expression in orthotopic tumour of nude mice, and CD271 expression and α-SMA expression in the stroma of gastric cancer, immunofluorescence microscopy was performed. The frozen sections or paraffin-blocked tissues were blocked with 3% BSA (diluted in PBS) for 30 min at room temperature. Orthotopic tumour was incubated with anti-F4/80 antibody (1 : 100; Abcam) for 2 h at room temperature and secondary antibody incubation (Alexa Fluor 647; 1 : 200; Abcam) was performed for 2 h at room temperature. Tissues were further incubated with anti-α-SMA antibody (FITC)- (1 : 150; Abcam) and anti-CD271 antibody- (1 : 100; Abcam) labelled Alexa Fluor 647 by Alexa Fluor 647 Monoclonal Antibody Labelling Kit (Life Technologies) for 2 h at room temperature and DAPI (Wako; 1 : 1000) for 30 min at room temperature. Tissues were viewed under a fluorescence microscope (Leica Digital Microscopy DMI 6000; Leica Microsystems, Heidelberg, Germany).

To examine the proportion of CD271-positive cells in cultured BM-SCs, the single-cell suspensions were washed in 500 μl PBS and resuspended in 100 μl per 10⁶ cells of PBS. The suspensions were stained with monoclonal CD271-FITC antibody (Miltenyi Biotech, Bergisch Gladbach, Germany) or anti-mouse IgG H&L Alexa Fluor 488 (Abcam, Cambridge, MA, USA) at 4 °C for 10 min. Stained cells were washed two times and resuspended in 500 μl PBS. The percentage of positive cells were calculated and compared with isotype-matched control-stained cells. Flow cytometry analysis was performed on a FACScan (BD LSR II; Becton Dickinson, San Diego, CA, USA). Furthermore, to examine the detailed quantification of the proportion of infiltrated bone marrow-derived cells, the orthotopic tumour cell suspensions were stained with anti-CD271 antibody- (Abcam) labelled Alexa Fluor 647 by Alexa Fluor 647 Monoclonal Antibody Labelling Kit (Life Technologies), and anti-F4/80 antibody (FITC) (Abcam) and anti-human/mouse CD11b (PE) antibody (Tonbo Bioscience, Burlingame, CA, USA) at 4 °C for 10 min.