Each sample was assayed in triplicate. The comparative CT method (ΔΔCT method) was used to determine the quantity
of the target sequences in TAM relative both to Mφ (calibrator) and to β-actin (an endogenous control). Relative expression levels were presented as the relative fold change and calculated using the formula: 2 -ΔΔCT= 2-(ΔCT (TAM) – ΔCT (Mφ) where each ΔCT =ΔCT target-ΔCT β-actin. Immunohistochemistry For exact identification of IL-10 or cathepsin B expression in TAMs, serial OSI-027 solubility dmso sections were used to examine the expression of IL-10, cathepsin B in TAMs. Samples were fixed in 4% formaldehyde in PBS (pH 7.2) and paraffin embedded. 4-μm thickness was cut from each paraffin block. After
Apoptosis inhibitor dewaxing and rehydration, the sections were microwaved for antigen retrieval in 10 mmol/liter citrate buffer (pH 6.0) for 10 min, and then allowed to cool for 1 hour at room temperature. Endogenous peroxidase activity was blocked with hydrogen peroxide; Nonspecific binding was blocked by preincubation with 10% goat serum Cilengitide clinical trial in PBS for 30 minutes at room temperature. Slides were incubated with the primary antibodies directed against monoclonal anti-human CD68 antibody (1:200 dilution, sc-20060, Santa Cruz, CA, USA), monoclonal anti-human IL-10 antibody (1:100 dilution, BA1201,Boster, WuHan, China) or polyclonal anti-human cathepsin B antibody (1:100 dilution, ab49232, Abcam, MA, USA). The results were visualized using the streptavidine-biotin immunoperoxidase detection kit and AEC chromogen (Maixin Bio, FuZhou, China) based on the manufacturer’s instruction. Positive cells stained red. The negative control involved omission of the primary antibody. Statistical analysis Statistical analysis software (Prism 5.0, GraphPad Software Inc, La Jolla, CA, USA and SPSS Version 13.0 software, SPSS Inc,
Chicago, IL, USA) was used to perform the analyses. Data are expressed as median (range). The Mann-Whitney aminophylline test was used for the comparison between TAM and normal macrophage. The correlation between IL-10 or cathepsin B expression and clinicopathologic factors was analyzed by Mann-Whitney test. Multivariate logistic regression was performed to evaluate the relationships between the pathological stage (with early and late stage as dependent variables) and covariates (age, sex, tobacco use, tumor histology and IL-10 expression in TAMs). For this analysis, the median value of IL-10 was chosen as the cut-off point for dividing the patients into the two groups. Two-tailed P value less than 0.05 was considered statistically significant. Results Patients characteristics The patient characteristics are described in Table 1. Patients (40 males and 23 females) had a mean age of 58.8 ± 1.1 years. Fifty-four patients had a smoking history, and forty-six were non-smokers.