Ivis spectrum imaging station

Cells treated with various drug combinations were washed with cold PBS and lysed in RIPA buffer containing 1mg ml⁻¹ DNAse (Roche Life Science, Indianapolis, IN) and supplemented with protease inhibitor cocktail (Roche Life Science, Indianapolis, IN). Cell lysates, stored frozen at −80 °C were resolved on 4–20% SDS-PAGE gels and transferred onto a nitrocellulose membrane. The membranes were first stained with anti-BIM (Cell Signaling Technology, Danvers, MA), and anti-HOXD10 (Santa Cruz Biotechnology, Dallas, TX) antibody cocktail followed by peroxidase conjugated-anti-rabbit antibody. ECL (Roche Life Science, Indianapolis, IN) was used to develop blots. Signal intensity was recorded on an IVIS Spectrum imaging station (Perkin Elmer, Hopkinton, MA). After documenting signal, the blot was subjected to a second round of immunostaining using an anti-actin-HRP antibody (Santa Cruz Biotechnology, Dallas, TX).

Cy5.5 labeled MN was administered by i.v. injection into a mouse with bilateral secondary tumors in axillary lymph nodes confirmed by bioluminescence imaging. A set of fluorescence images was collected using an IVIS Spectrum imaging station (Perkin Elmer, Waltham, MA) for the reconstruction of 3D fluorescence imaging tomography (3D-FLIT) using Living Image software (ver. 4.4, Perkin Elmer, Waltham, MA). The excitation wavelength was fixed at 675nm and the emission wavelength was 720nm. Overall, 18 fluorescence images were processed to reconstruct 3D-FLIT for the illustration of fluorescence sources in the body. From these, 2-D images were extracted in the coronal and transaxial planes.