Resveratrol in IL-2 Therapy against Melanoma Figure 4. Histological studies of the lung and the liver for inflammatory cell infiltration. Lungs and livers from VLS mice were harvested and preserved in 10% formalin solution. Sections were stained with hematoxylin and eosin. The level of perivascular infiltration was determined by counting the number of cells infiltrating around a venule. The data depicts the mean 6SEM of sections from four individual mice. A. The level of perivascular infiltration in lungs. B. The level of perivascular infiltration in livers. C. Statistic analysis of the infiltration depicts p.0.05 between IL-2 and IL-2+Resveratrol groups. Original magnification, 6200. doi:10.1371/journal.pone.0035650.g004 positive stained endothelial cells were counted in 5 vessels of each slide and the data were expressed as mean 6SE of apoptotic cells per vessel. Histological Analysis of cell infiltration and measurement of serum TNF-a The lungs and livers were fixed in 10% formalin solution. The organs were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Perivascular infiltration was scaled by counting the number of lymphocytes infiltrating around the vessel and averaging the minimum and maximum range for each group. Five samples per mouse were analyzed, and a minimum of four mice were included. Sera were prepared for detection of TNF-a by sandwich ELISA using TNF-a ELISA kit (Biolegend). Electron microscopic studies of vascular injury Tissue samples were fixed in 5% glutaldehyde/4.4% formalde- hyde/2.75% picric acid in 0.05 M sodium cacodylate buffer, pH 7.4, washed in a sodium cacodylate buffer, postfixed in osmium tetroxide, embedded in Polybed 812 resin (Polysciences, Warrington, PA) and studied with an electron microscope. [3H]-thymidine incorporation assay LAK cells generated from IL-2 treated mice were cultured in complete RPMI 1640 medium in a 96-well round bottom plate. 3 [ H]-thymidine (1 mCi/well) was added to the culture. In some groups, LAK cells were incubated with MDSC at 10:1 ratio 3 (LAK:MDSC) for 2 h before adding [ H]-thymidine. At 16 h, cultures were harvested using a cell harvester and the radioactivity incorporated in the cells was measured in a beta counter (Microbeta counter, Perkin Elmer). Quantitative real-time PCR (QPCR) Total RNAs were prepared and reversely transcribed to cDNA using miRNeasy Mini Kit and miScript Reverse Transcription Kit (Qiagen). FoxO1, arginase 1 (Arg1), aryl hydrocarbon receptor (AhR), and 18S RNA (18S) were amplified using miScript SYBR Green PCR kit (Qiagen) in a StepOnePlus PCR amplifier (ABI). The relative gene expression level was normalized to the endogenous control 18S. The primer sequences were listed below. FoxO1 F: TGT TAG GCC CAC CAC ACG AGT TTA; FoxO1 R: GGT GAA AGC AAA GGG CTC CAA TGT; Arg1 F: ACG GCA GTG GCT TTA ATT GGC; Arg1 R: TCT GTC TGC TTT GCT GTG ATG CC; NOS2 F: TGC CAC CAA CAA TGG CAA CAT C; AhR R: TTG AAG TCA ACC TCA CCA GCA GC; 18S F: AAA GGA ATT GAC GGA AGG GCA CC; 18S R: AAC TAA GAA CGG CCA TGC ACC AC. Statistical analysis Data were presented as mean 6SEM and analyzed for significance using non-paired Student’s t-test. In case of multiple testing, One-way ANOVA test followed by Newman-Keuls PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35650