Moma 2

Tumors isolated from mice were immersed in 4% PFA at 4°C for 2 h, immersed into 30% sucrose solution at 4°C overnight, embedded in tissue fixation solution (OCT), and rapidly frozen at −80°C. The neutrophil (Neu, Santa Cruz Biotech., SC-71674), S. Typhimurium (SL, Bio-Rad, 8,209–4,006), monocyte/macrophage (MOMA-2, Santa Cruz Biotech., SC-59332), and CD206 (M2 macrophage marker, Santa Cruz Biotech., SC-34577) in the tumor tissue sections of mice were analyzed by a standard immunofluorescence staining protocol. Images were obtained under an LSM510 fluorescence microscope (ZEISS, Germany) and processed using laser scanning microscopy (LSM) image software.

Immunohistochemistry (IHC) was essentially as we described in refs. [15 (link), 16 (link)]. Briefly, sections were fixed in acetone and processed for immunostaining for MOMA-2 (#sc-59332, Santa Cruz Biotechnology, TX) followed by incubation with biotinylated rabbit anti-rat antibody (Dako). After treatment with secondary antibodies sections were incubated with alkaline phosphatase-conjugated streptavidin (Dako). Counterstaining was with hematoxylin. Negative controls were performed by substituting the primary antibody with non-immune IgG from the same species and at the same concentration. Under these conditions, nonspecific immunostaining was not detected. Stained areas were captured (Micropublisher 3.3 Megapixel Cooled CCD Color Digital Camera) and measured (NIS Elements D). Either MOMA-2 or the rabbit polyclonal antibody to AIA31240 (Accurate Chemical and Science Corp.) was used to identify macrophages in co-localization immunofluorescence staining. Secondary antibody was Alexa Fluor-488 goat anti-rabbit (#A11008, Invitrogen, for AIA31240) or Alexa Fluor-488 anti-rat (#4416, Cell Signaling, for MOMA-2). Immunostaining for the Ki67 antigen was performed by incubating acetone-fixed frozen sections with an anti-Ki67 antibody (#ab66155, Abcam; 1:100 dilution) overnight at 4°C followed by anti-rabbit IgG Alexa Fluor-555 (#4413, Cell Signaling) at 1:1,000 dilution for 1 h.