Internalization and specificity of PhD-targeted peptide phage in CP cells in culture. TR-CSFB cells were incubated with untargeted or targeted phage (5 × 1010pfu/ml) for 2 h at 37C, acid washed to remove any particles on the cell surface, fixed and then immunostained with antibodies against M13 phage and subsequently Alexa594-labeled goat anti rabbit antibodies. Little internalization is observed when the cells were treated with control phage (Panel A) and while cells tended to clump together upon staining, significant intracellular immunoreactivity was nevertheless observed in TR-CSFB cells after incubation with Peptide B (Panel B), Peptide C (Panel C) or Peptide D (Panel D) targeting of phage. M13 phage: red; Cell nuclei (DAPI): white. To demonstrate that internalization was ligand-mediated, TR-CSFB cells were incubated with targeted phage (1 × 1010pfu/ml) for 2 h at 37C in the absence (Panel A) or presence of an excess (200 ng) of Peptide C (Panel E) or Peptide D (Panel F). M13 phage: red; Cell nuclei (DAPI): white.