FeCo/GC Macrophage Imaging Figure 3. Immunostaining of macrophages and FeCo/GC. (A) Immunohistochemical staining showed macrophages (by anti-Mac3 antibody) infiltrating the neointima of the ligated left carotid artery. (B) Immunohistochemical staining for smooth muscle cells (by anti-SMA antibody). (C) Quantitative analysis of proportion of neointima composed of macrophages and smooth muscle cells. (D) Immunofluorescent staining demonstrated macrophages (green), FeCo/GC-Cy5.5 (red), nuclei (blue), and combined staining with strong colocalization (yellow), particularly near the luminal border (arrow). (E) Immunofluorescent staining of the macrophage-rich liver also demonstrated strong colocalization (yellow) of FeCo/GC and macrophages. (F) By contrast, the macrophage-poor heart tissue showed scant FeCo/GC uptake, with the few spots of uptake colocalizing with macrophages (arrows). doi:10.1371/journal.pone.0014523.g003 [27]. At day 5 of the STZ injections, serum glucose was measured from tail vein blood using a glucometer. If the glucose level was below 200 mg/dL, animals were injected with STZ for 3 additional days. Two weeks after the initiation of STZ injection, the left common carotid artery was ligated below the bifurcation with the use of 5-0 silk ligature (Ethicon) under 2% inhaled isoflurane [28]. Sham operation was performed by passing the suture under the left carotid artery without constricting the artery. The wound was closed by suture and the animals were allowed to recover on a warming blanket. FeCo/GC synthesis The 7 nm FeCo/GC were synthesized as previously described [23,24], then sonicated with 1 mg/mL phospholipid-polyethylene glycol (PL-PEG) in water for one hour. To obtain singly suspended FeCo/GC, the solution was centrifuged for 6 hours at 24,0006 g. Excess PL-PEG was removed by filtration and UV-vis was used to determine nanocrystal and metal concentration. For fluorescence imaging experiments the PL-PEG used was DSPE-PEG5000-NH2 (NOF corp.). For fluorescent dye attach- ment, Cy5.5-NHS ester was mixed with FeCo/GCPL-PEG-NH2 in phosphate buffered saline. Dye that was not attached to the nanocrystal was removed by filtration. Samples prepared for in vivo MRI imaging at 3 T were functionalized using DSPE-mPEG5000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine–N-[methoxy(- polyethylene glycol)5000], Laysan Bio). In vivo uptake and fluorescence imaging To verify in vivo uptake of Fe/Co-GC in the carotid macrophages, Cy5.5 was conjugated to FeCo/GC, allowing fluorescence imaging and microscopy. FeCo/GC-Cy5.5 (8 nmol of Cy5.5/mouse; 32.14 mg Fe/mouse) was injected intravenously via tail vein into mice two weeks after carotid ligation (n = 6) or sham operation (n = 2). These mice were imaged at 48 hours using the MaestroTM in-vivo imaging system (Cri, Woburn, MA). Under inhalational anesthesia (2% isoflurane), left and right carotid arteries were surgically exposed and in situ florescence imaging was performed. Then, the carotid arteries were carefully removed en bloc followed by ex vivo fluorescence imaging and immunohisto- chemistry (see below). In vivo MRI To study noninvasive imaging of FeCo/GC by MRI, ligated mice (n = 6) were injected with FeCo/GC (270 mg Fe/mouse) and imaged serially at 48 and 72 hours on a whole-body 3T MRI PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e14523