The fluorophore excitation and emission filter sets used were ��excitation selleck chemicals = 445�C490 nm and ��emission = 515�C575 nm. The fluorescence images shown are real-time unprocessed images. Fluorescent signals within the liver region were normalized to the background fluorescence taken over the same region from noninjected animals. Detection of GFP+ MSC by immunohistochemistry. Sections (5 ��m) were obtained from formalin fixed and paraffin-embedded Matrigel plugs implanted subcutaneously with MSCGFP or MSCsIGFIR cells. The sections were deparaffinized and immunostained with a rabbit anti-GFP antibody and an Alexa Fluor 568 goat anti-rabbit IgG (both from Molecular Probes, Invitrogen Canada), both used at a dilution of 1:200. Images were obtained using a Zeiss LSM 510 Meta confocal imaging station and the Zeiss LSM Image Browser (Carl Zeiss Canada, Toronto, Canada).

Terminal deoxynucleotidyl transferase�Cmediated nick end�Clabeling assay. To measure the extent of apoptosis within micrometastatic lesions, the livers were removed and snap frozen 6 days postintrasplenic/portal injection of GFP-tagged H-59 cells and 8 ��m cryostat sections prepared. Apoptotic cells were labeled by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay, using the in situ cell death detection kit, TMR red (Roche Diagnostics, Laval, Quebec), according to the manufacturer’s instructions. Nuclei were stained with 4��,6-diamidino-2-phenylindole and the sections mounted with the ProLong Gold antifade reagent (Invitrogen), visualized with the LSM 510 Meta confocal microscope and images acquired and analyzed with the Zeiss LSM Image Browser program.

Microvessel density and Ki67 staining. To measure tumor-induced angiogenesis, the mice were inoculated with 105 tumor cells by the intrasplenic/portal route, killed 6 days later, and the livers perfused via the portal vein with a solution of 4% paraformaldehyde in phosphate-buffered saline, excised, and processed, as we previously described,32,39 before the preparation of 8 ��m cryostat sections. To stain in
gut barrier dysfunction is well documented in animal models of short bowel syndrome (SBS) after massive small bowel resection. This is evidenced by translocation of luminal microbes to mesenteric lymph nodes (MLN) and subsequently to blood and peripheral organs such as liver and spleen (3, 9, Entinostat 26, 31). This phenomenon may contribute to the common incidence of infections due to gut-derived microorganisms in SBS patients requiring parenteral nutrition (28, 36). Our recent studies demonstrate a high incidence of detectable flagellin and lipopolysaccharide (LPS) in the serum of adults with SBS and elevated specific immunoglobulins (Ig) against these gram-negative bacterial antigens (44).