A three-color immunocytochemistry analysis (30) was adopted for immunofluorescence characterization of CTCs captured from blood samples collected from patients with NSCLC. The cells captured on Tz-grafted SiNWS were incubated with 0.05% Triton X-100 [in PBS (200 μl)] for 10 min. The captured cells were incubated overnight with a mixture of primary antibodies including Pan-CK antibody [rabbit, polyclonal, 1:100 (v/v); Dako] and anti-CD45 antibody [F10-89-4] [mouse, monoclonal, 1:400 (v/v); Abcam] in a PBS solution (200 μl) containing 2% normal donkey serum (Jackson ImmunoResearch) at 4°C. After washing with PBS three times, the captured cells were further incubated at room temperature for 1 hour with a mixture of secondary antibodies including donkey anti-rabbit IgG (H+L) [Alexa Fluor 488, 1:500 (v/v); Invitrogen] and donkey anti-mouse IgG (H+L) [Alexa Fluor 647, 1:500 (v/v); Invitrogen] in a PBS solution (200 μl) containing 2% donkey serum. After washing with PBS three times, the cells were treated with ProLong Gold Antifade Mountant with DAPI (Invitrogen). The substrates were then imaged with a fluorescence microscope (Nikon 90i). The captured CTCs were differentiated from background WBCs according to their unique staining pattern (CK+/CD45/DAPI+) and intact nuclear morphology (WBCs were stained CK/CD45+/DAPI+).

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