Alexa fluor 488 goat anti mouse igg2b secondary antibody

Two slides from patient UM-001 were stained with pan-cytokeratin (CK) and CD45 antibodies as described above with DAPI^{24 (link)} complemented with a monoclonal EpCAM antibody (1:250, 324202, Biolegend, San Diego, CA; Alexa Fluor® 488 goat anti-mouse IgG2b secondary antibody, 1:500, A21141, Invitrogen, Carlsbad, CA) at Epic Sciences. CTCs were identified with standard image analysis protocols^{25 (link)–35 (link)}.

Lipo/siRNA complexes (100pmols of siRNA) marked with rhodamine were incubated with PBMC cultured in 48-well culture plates for 4 and a half hours. Paraformaldehyde-fixed cell suspensions were located on microscope slides as described above. For comparing Lyp protein expression among CD3⁺ and CD3^- cells the following antibodies were used: monoclonal mouse IgG_2B anti-human Lyp antibody (Clone #340113, 1:100; R&D Systems, O/N at 4°C), Alexa fluor 488 goat anti-mouse IgG2b secondary antibody (Invitrogen, 1:800 incubated for 1 hour at RT); mouse anti-human CD3 primary antibody (BD Biosciences 1:30, incubated for 1 hour at RT); Alexa Fluor 647 goat anti-mouse IgG1 secondary antibody (Invitrogen, 1:800 incubated for 1 hour at RT). Nuclei were counterstained with 1 μg/ml Hoechst 33342 (Invitrogen) as above described.