Molecular Imaging of Fibrosis Figure 6. Imaging of lung fibrosis. A: Paraffin embedded sections of lungs from a bleomycin-treated mice were incubated with B-collagelin and detected using HRP-coupled streptavidin. Sections were counter-stained with hematoxylin. Contiguous serial sections were stained with picrosirius red. Representative images are shown. B: Whole body planar scintigraphy of a bleomycin-treated mouse (left) and a control mouse (right). Representative images acquired one hour after the injection of 99mTc-collagelin are shown. C: Corresponding lung autoradiography and histology (picrosirius staining) showing the colocalisation of 99mTc-collagelin with fibrosis. D: Lung autoradiogaphy and histology from a mice treated with the control 99mTc-Pc. doi:10.1371/journal.pone.0005585.g006 Pc (SGSGCGPQGARGLTGASAGGPC), biotinylated at the N– terminus (Mimotopes, Clayton Victoria, Australia) were used for control experiments. Binding experiments Surface plasmon resonance. 9O12.2 IgG was injected over biotin-peptides coupled to an SA-sensorchip in a Biacore 2000 instrument. Kinetic constants (kon, koff) were deduced at different IgG concentrations (from 1.25 to 10 mg.mL21), and the dissociation constant KD = koff/kon was calculated. In some experiments, the peptide was reduced by DTT on the sensor chip before the IgG was injected. Competition experiments were performed by mixing the IgG with recombinant soluble GPVI (25 mg.mL21). Collagen binding to the peptide was analyzed by injecting type-I collagen (200 mg.mL21) onto the peptide-coated sensorchip. Conversely, biotin-peptides (125, 250, 500 and 1000 mg.mL21) were perfused over fibrillar collagen immobilized on a CM5-sensorchip. Solid phase assays. Biotin-peptides (50 mg.mL21) were incubated on microtitration wells, coated with 1 mg of type-I collagen, convulxin, collagen related peptides, bovine fibrinogen, human fibronectin, rat vitronectin, murine laminin or bovine serum albumin. Bound peptides were detected using HRP-coupled extravidin and the HRP substrate O-phenyl-dianisidine. Histochemical analysis Frozen sections (8 mm) of rat thoracic aorta embedded in tissue- Tek OCT were incubated with biotin-peptides (200 mg/mL) revealed using a HRP-coupled streptavidin. Sections were counter-stained with hematoxylin-eosin (Merck). Collagen was stained using picrosirius red. Sections (20 mM) of rat tail tendon were processed as above. Ex vivo and in vivo imaging with radiolabeled collagelin Biotin-collagelin and biotin-control peptide, Pc, were radiola- beled as follows. Streptavidin was dissolved in 0.01% (v/v) acetic acid at a concentration of 1 mg.mL21. Reactants were mixed for 1 min in the following order: first 10 mL of streptavidin followed by 4 mL of stannous pyrophosphate, 2 mL of KBH4 (10 mg.mL21 in 0.1 N NaOH) and 740 MBq of Tc-99m in 50–100 mL. Thirty min later, the solution was ready for injection. Radiolabeling efficiency was measured by thin-layer chromatography, using the classical methyl-setone method High efficiency of labeling was obtained, superior to 96% at 30 min. Labeled streptavidin was mixed with B-collagelin in a molar ratio of one to four immediately before injection. B-collagelin was directly labeled using the same procedure as above: 10 mL of peptide [2 nmol in 0.01% (v/v) acetic acid] was mixed with 740 MBq of Tc-99m for 30 min. High efficiency of labeling was obtained, superior to 96% at 30 min. In this latter case, Tc-99m was covalently complexed by disulfide bonds, ensuring the high stability of the radiolabeling For control analyses, B-Pc was used, after radiolabeling with 99mTc-streptavidin or directly by Tc-99m, using the above- described procedures. The labeling of streptavidin and peptides was stable for at least three hours, and then decreased with 40 to 50% free technetium being detectable at 6 hours. Labeled products were injected intravenously (iv) at the dose of 70 MBq/rat. Myocardial infarction of the left ventricle was induced in rats as follows. Male Wistar rats (CERJ, France) were housed in a controlled environment and fed with a standard rat chow. Myocardial infarction of the left ventricle was induced by ligature of the left descending coronary artery performed under general PLoS ONE | www.plosone.org 8 May 2009 | Volume 4 | Issue 5 | e5585