Internalization of antibody coated Dynabeads Protein A into 4T1-HER2 cells. The beads were coated with the first antibody (1) and incubated with 4T1-HER2 cells. Following washing, the cells were incubated with the second antibody (2) and analyzed by confocal immunofluorescence microscopy. Beads labeled with (1) are located intracellular, while beads labeled with (1) and (2) are located extracellular. Non coated beads showed no background fluorescence (I) and were efficiently coated with Trastuzumab-Alexa488. On bead-coating with Trastuzumab or Trastuzumab-Alexa488 (II, III) some beads were located in the cell (marked with white arrowheads). Some beads remained outside the cells (marked with black arrowheads). Presence of bead fluorescence was analyzed in image stacks of at least 5 μm thickness to exclude false negatives (Additional file 4).