Fig. 3

The analysis of humanized antibody variant CA9hu-1(HC4LC4) for its capacity to internalize into cancer cells. Visualization by confocal microscopy revealed punctuated intracellular staining signal after acid strip indicating internalization. Cells were incubated with MAb at 4 °C for 30 min and then in parallel at 4 °C (to prevent internalization) and 37 °C (to trigger internalization) for 3 h. After fixation with methanol, the cells were stained with anti-human Alexa Fluor® 488-conjugated antibody in order to visualize the MAbs. Acid strip before fixation was used to remove the membrane-bound antibody. Scale bar = 20 μm