Anti c myc antibody clone 9e10

For immunocytochemistry (ICC), 50 000 target cells (HBVP, PBVP, hMscTert, ASF2, HBMEC and HMEC-1) were seeded in each well of an 8-well Nunc Lab-Tek Chamber Slide System and incubated overnight for attachment. The cells were rinsed with dPBS, fixed in 2% PFA for 15 min at RT and subsequently blocked in 2% (w/v) MPBS for 1.5 h at RT. For staining on HBVP only, the primary antibodies were diluted to a final concentration of 40 μg/ml in 2% MPBS and incubated overnight at 4°C. The next day, the wells were washed 2 × 5 min in PBS and incubated with a secondary anti c-Myc antibody (clone 9E10, Sigma-Aldrich) diluted 1:500 in 2% (w/v) MPBS, for 2 h at RT. The cells were washed 3 × 5 min in PBS and the secondary antibody was detected by an Alexa 488 conjugated Goat anti Mouse IgG antibody (Thermo Fisher Scientific). For assessment of antibody specificity though ICC on different cell types, the primary antibodies were diluted to a final concentration of 60 μg/ml in 2% MPBS and incubated overnight at 4°C. The wells were washed 2 × 5 min in PBS and the primary antibodies were detected by a monoclonal mouse Anti-c-Myc-Cy3 antibody (clone 9E10, Sigma-Aldrich) diluted 1:100. For both experiments, cell nuclei were stained using VECTASHIELD Mounting Medium with DAPI (Vector Labs, USA). Fluorescent images were obtained with a Leica DMI3000 B inverted microscope (Leica Microsystems, Germany).