Immunofluorescence Analysis of Cell Markers

The immunofluorescence analysis was carried out according to previously published methodology [50 (link)]. Anti-68 antibody (Santa Cruz Biotechnology) and anti-iNOS antibody (Santa Cruz Biotechnology) and anti-Arg-1 antibody (Santa Cruz Biotechnology) were incubated in a humidified chamber at 37 °C O/N on the sections. After washing with PBS, sections were incubated for 1 h at 37 °C with secondary antibodies, TEXAS RED-conjugated anti-rabbit Alexa Fluor-594 (Molecular Probes, Eugene, OR, USA) and FITC-conjugated anti-mouse Alexa Fluor-488 (Molecular Probes, Eugene, OR, USA). Nuclei were stained by adding 2 μg/mL 40,60-diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt, Germany) in PBS. Slides were then washed with PBS and incubated with a secondary antibody. Specific labeling was identified with an avidin–biotin-peroxidase complex and a biotin-conjugated goat anti-rabbit immunoglobulin G (Vector Lab, Milan, Italy) [51 (link)]. Stained sections were observed using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy). Each specimen was observed in five random visual fields and the average number of double-positive cells in each specimen was calculated [19 (link)].