Grimm et al. BMC Cancer 2010, 10:82 http://www.biomedcentral.com/1471-2407/10/82 PCR-analysis. Paraffin sections were evaluated using hematoxylin and eosin. The rest of the liver tissue was either snap-frozen and stored at -80°C for RNA extrac- tion and Real Time PCR analysis or fixed in acetone for Cryostat sections. Monoclonal antibodies (mAb) were purchased as fol- lows: anti-CD4-mAb (MCA 1767), anti-CD8-mAb (MCA 609G), anti-CD25-mAb (14-0251), anti-Foxp3- mAb (14-5773) from eBioscience (San Diego, CA, USA), anti-FAS-L-mAb (ab21233) and anti-FAS-mAb (321-335 Cat. No. PC69) from Calbiochem (Darmstadt, Germany). Cy3 conjugated anti-CD4-mAb and anti- CD8-mAb were purchased from Linaris (Wertheim, Germany). Isotype-matched mAbs or purified IgG1 (MCA1211, Serotec, Oxford, England) and controls for residual endogenous peroxidase activity were included in each experiment. The analysis of single staining was performed for CD4, Foxp3, CD8, CD25, FAS, and FAS-L. Immunohisto- chemical double staining was carried out for CD4 and CD8 following standard staining procedures. For immu- nohistochemical double staining, antibodies bound dur- ing the first staining step were eluted using LIN-Block (RAG0149LK) (Linaris, Wertheim, Germany) according to the manufacturer’s instructions, and the slides were subsequently incubated with the second primary mAb diluted in TBS plus 0.5% BSA for 20 min at 37°C fol- lowed by AP-conjugated secondary antibody and devel- opment with Vector Blue (SK-5300) (Linaris) for 30 min and counterstaining with hemalaun for 1 minute. The sequential immunofluorescence double staining (coexpression) was detected with Foxp3+ and CD4+ and FAS+ with CD8+ cells. The slides were incubated with the primary antibody or control antibody diluted in TBS plus 0.5% bovine serum albumin (BSA) overnight at 4°C in a humidified chamber and with secondary FITC-con- jugated (fluorescein isothiocyanate) antibody for 30 min- utes at room temperature in a humidified chamber. The slides were blocked with 10% normal rat serum diluted in TBS and incubated with the Cy3 conjugated primary antibody diluted in TBS plus 0.5% BSA overnight at 4°C in a humidified chamber. Slides were counterstained with DAPI (4’,6-diamidino-2-phenylindole) if necessary (Sigma-Aldrich, Steinheim, Germany). The quantification of each immunohistochemical staining was done by cell counting in six individual representative high power fields (× 400). Likewise, the evaluation of immunofluorescent double staining was performed by counting CD4+ or CD8+ cells in six high power fields (×400) together with Foxp3+ or FAS+ cells in cryostat sections. The proportion of Foxp3 positivity in counted CD4+ and the proportion of FAS positivity in counted CD8+ cells were expressed in percentages. The result of the staining was expressed in percentages Page 3 of 11 (%) positivity. Results were examined by two blinded and unbiased colleagues experienced in gastrointestinal pathology and histology. RNA extraction RNA was extracted using an RNA extraction kit (Qia- gen, Hilden, Germany) from at least 10 mg of homoge- nized tumor tissue. After homogenization DEPC-75% ethanol was added to the lysate to provide ideal binding conditions. The lysate was then loaded onto the RNeasy silica membrane ("RNeasy Mini spin column”). After binding of RNA all contaminants, including genomic DNA, were efficiently washed out. Pure, concentrated RNA was eluted in water and stored at -70°C until further analyses. The amount of total RNA was deter- mined by measuring absorbance at 260 nm. The purity of the total RNA was established by confirming that the 260 nm: 280 nm ratio was within a 1.8-2.0 range, indi- cating that the RNA preparations were free of protein contaminants. RT-PCR for cytokine genes in metastatic tumor specimens mRNA expression of representative surface molecules and cytokines (CD4, CD8, CD25, Foxp3, IL-10, TGF-b, TNF-a, IFN-g, FAS, and FAS-L) was analyzed in meta- static tumor specimens by Real Time PCR. Experiments were done 4-6 times per animal. RNA was extracted as described above. cDNA was prepared using 2 μg of heat-denatured RNA. Primer sets from Quiagen (Hilden, Germany) were used for analysis. Optimum primer con- centration was determined by titration. Real Time quan- titative PCR was performed in a two-step RT-PCR using SYBR-Green PCR Master Mix (PE Biosystems, Foster City, CA) with 100 ng cDNA and 300 nM of primers in a total reaction volume of 50 μl. PCR thermal cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. Gene specific products were continuously measured by an ABI PRISM 7700 sequence detector (Applied Biosys- tems, Foster City, CA) and relative quantification was performed following the manufacturer’s instructions. In situ detection of apoptosis To identify cells with fragmented DNA we used a noniso- topic DNAend-labeling in situ technique, employing digoxigenin-dNTP and terminal transferase (ApopTag® Fluorescein in Situ Apoptosis Kit, Chemicon, Planegg- Muenchen, Germany). Briefly, sections were post-fixed and equilibrated in terminal transferase buffer before the addition of reaction buffer containing digoxigenin-dNTP oligonucleotide. The digoxigenin-dNTP-containing oligo- nucleotide extensions were detected by anti-digoxigenin- fluorescein conjugate diluted in a blocking agent, followed by blocking with 10% normal rat serum diluted in TBS