Identifying a Cardiac PTD Figure 4. Internalization and quantification of transduction by CTP-6CF in cardiac tissue in vivo. Cross-sections of mouse heart were stained for actin (blue) and laminin (red). Confocal microscopy showed co-localization of CTP-6CF (a) with actin (b) but not laminin (c) as seen in the color-merged micrograph (d). FITC fluorescence from non-overlapping heart micrographs from mice injected with CTP-6CF (n = 3) or PBS (n = 3) was quantified and expressed as a percentage of total area calculated from staining for actin (Fig. 4e; error bars represent standard error of the mean). CTP-6CF – green; Actin – blue; Laminin – red. Scale bars represent 100 uM. doi:10.1371/journal.pone.0012252.g004 To confirm further the ability of CTP to transduce cardiac tissue in vivo, the peptide was coupled to fluospheres that allow for analysis of localization by whole animal imaging. Balb/c mice were injected intracardiac with 40 nm neutravidin-labeled fluo- spheres alone, CTP-biotin and CON-biotin labeled with these fluospheres. Mouse imaging was performed at baseline and 30, 60, 120 and 180 minutes. CTP+fluospheres were retained in the heart, as opposed to fluospheres alone or CON+fluospheres, which dissipated immediately after injection. CTP+fluospheres could still be found localized to the heart at 3 hours post-injection (Fig. 7). To determine the relative efficiency as well as specificity of transduction of cardiac tissue by CTP, the transduction ability of PLoS ONE | www.plosone.org 6 August 2010 | Volume 5 | Issue 8 | e12252