Pe anti mouse igg1 antibody

In order to confirm the purity of isolated cells, we used fluorescence-conjugated antibodies targeting the proteins expressed on CTCs. Cell fixation, permeabilization, and blocking were done before immunostaining process.[8 ] Rinsing the cells in PBS is required between each steps. First of all, the isolated cells were fixed and permeablized using 4% paraformaldehyde and Tritonx 0.2%, respectively. Next, nonspecific binding sites were blocked using BSA 1% for 1 h at 37°C. Then, the cells were labeled with FITC-epithelial cell adhesion molecules (EpCAM) antibody (1:500 FITC anti-human CD326 Antibody, BioLegend) and β4 integrin antibody (0.5/100 microliter PBS, Anti-Integrin β4 Antibody, clone ASC-8, Sigma-Aldrich) overnight at 4°C. Then the cells were incubated with PE-IgG1 antibody (PE anti-mouse IgG1 Antibody, BioLegend) as a secondary antibody for 40 min at 37°C. Furthermore, in order to label double-stranded DNA and thus visualizing the nuclei, the cells were stained by Hoechst®33342 dye at 37°C for 20 min (5 μg/ml). In addition, the cells were labeled with using pancytokeratin antibody (1:500 Alexa Fluor® 488 anti-Cytokeratin, BioLegend), another CTC marker, following with β4-integrin staining. Images were captured using an inverted fluorescent microscope (Leica Microscope, USA) at ×20 magnification.