Figure 2: In vivo binding of SPs-displaying phages to the mouse carotid artery endothelium.
(a) Validation of individual selected phage clones in the endothelium of mice. The control phages or six SPs-displaying phages (2 × 1011 pfu) were injected intravenously into C57BL/6 mice (n = 3) at 3 days after LCA ligation, and allowed to circulate for 10 min. Both carotid arteries were removed and the level of endothelium-bound phage was compared by en face staining. Carotid arteries were fixed and bound phages were stained using an anti-M13 bacteriophage antibody; nuclei were stained with DAPI (red: phage, blue: nuclei) (magnification, ×400; scale bars, 10 μm). Representative images are shown. Relative mean fluorescence intensity calculated using Image J for phage binding (red). *Significant difference (RCA vs. LCA, p < 0.05). (b) Localization of the phages attached on the endothelium. Phages attached to the luminal surface of the endothelium were verified by confocal microscopy z-stack imaging (red: phage, blue: nuclei, green: auto fluorescent of elastic lamina) (magnification, ×400; scale bars, 10 μm). Representative images are shown. (c) Bio-distribution of SPs-displaying phages in various organs including carotid arteries were quantified by titrating phage plaque numbers in the tissues obtained from the mice 10 min after the injection of two phages. Bars represent the titers of phages from the third round of amplification in various tissues and are presented as means ± SEM of three independent experiments. *Significant difference compared with other organs, with the exception of the liver and spleen (p < 0.05).
