Molecular imaging software

Kidney tissue was homogenized with protease inhibitors in ice-cold RIPA buffer. For electrophoresis, antibodies were used as the primary antibodies. Anti-rabbit or anti-mouse IgG antibodies conjugated to HRP were used as the secondary antibodies. Membrane-bound HRP-labeled protein bands were monitored with chemiluminescent reagents, and signals were detected using Kodak Molecular Imaging Software (Eastman Kodak Company, Rochester, NY, USA).

For western blot analysis, the cells or lung tissue (30–50 mg) was prepared into homogenate samples and lysed in a RIPA-lysis buffer with protease inhibitors, followed by centrifugation (12,000× g, 20 min) and the protein concentration determined by a Bio-Rad protein assay kit (BioRad, Hercules, CA, USA). Equal amounts of protein were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresed proteins were transferred onto PVDF membranes. After blocking, the membranes were incubated with primary antibody (1:2000 dilution). Appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma, St. Louis, MO, USA) were applied and the signals were detected using the enhanced chemiluminescent method (Amersham International plc., Buckinghamshire, UK). The western blot analysis was performed using Kodak Molecular Imaging Software (Eastman Kodak Company, Rochester, NY, USA). Strip and reprobe blots by stripping buffer (62.5 mM Tris-HCl (pH 6.7), 2% SDS, 100 mM β-mercaptoethanol) in a sealed bag at 50 °C for 30 min, followed by two washes in PBS-Tween for 10 min each. Membranes were then stripped and could be reprobed, beginning with the blocking step [17 (link)].

The activities of MMP-2 and -9 in both the medium and serum were assayed by gelatin zymography according to the protocol developed by Kleiner and Stetler-Stevenson with minor modifications^{41 (link)}. Briefly, the culture medium was collected and electrophoresed in an 8% SDS-PAGE gel containing 0.1% gelatin. In animal studies, the serum samples of the mice were pooled because of the limited serum volume and were diluted 1:40 with PBS immediately before the assay. After electrophoresis, gels were washed at room temperature with 2.5% (v/v) Triton X-100 and subsequently transferred to reaction buffer for enzymatic reactions containing 1% NaN₃, 10 mM CaCl₂ and 40 mM Tris-HCl, pH 8.0, at 37 °C with shaking overnight. Finally, the gel was stained with 0.25% (w/v) Coomassie blue in 10% acetic acid (v/v) and 20% methanol (v/v) and destained in 10% acetic acid (v/v) and 40% methanol (v/v). The relative MMP-2 and -9 activities were quantified by Kodak Molecular Imaging software (version 4.0.5, Eastman Kodak Company, Rochester, NY) and represented by their relative intensities.

Liver tissues were homogenized in lysis buffer (0.6% NP-40, 150 mM NaCl, 10 mM HEPES (pH 7.9), 1 mM EDTA, and 0.5 mM PMSF) at 4°C. Fifty microgrammes of protein subjected to 10 % SDS-PAGE was transferred to polyvinylidene fluoride (PVDF) membrane (NEN Life Science, Boston, MA), blot immunodetected with enhanced chemiluminescence (ECL) Western blot kit (Amersham International, Amersham, UK) in which goat anti-mouse COX-2, nuclear factor-kappaB (NF-κB), inducible nitric oxide synthase (iNOS) and β-actin antiserum served as primary antibody (Millipore, MA) and goat anti-rabbit IgG-biotinylated species-specific whole antibody (Calbiochem) was used as secondary antibody. Blots were quantified by relative intensity compared to control with Kodak Molecular Imaging Software (Version 4.0.5, Eastman Kodak Company, Rochester, NY), represented in relative intensities.

In vivo fluorescence imaging was performed using a Kodak In-Vivo FX Pro Imaging System (Kodak, Woodbridge, CT, USA) and analyzed with Kodak Molecular Imaging Software (Kodak). A filter set (excitation wavelength, 610 nm; emission wavelength, 700 nm) was used for achieving NIR fluorescence in vivo. Identical illumination settings were used to obtain all images.
For the experiment, mice were injected via the tail vein with 0.5 nmol of probe. Mice in the Ab-FL-Cy5.5 group were injected with Ab-FL-Cy5.5 probe, and mice in the IgG-Cy5.5 group were injected with IgG-Cy5.5, which was used to evaluate the non-specific binding effects of antibodies. NIR fluorescence images were acquired at 4, 12, and 36 h post injection (p.i.). The mice were subsequently sacrificed at 36 h p.i. The tumor and major organs were dissected for evaluation with ex vivo fluorescence imaging.