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The sections were submerged in citrate antigenic retrieval buffer and subjected to high pressure for antigenic retrieval. Then slides were incubated with EBI3 (1 : 200 dilution, sc-32868, Santa Cruz Biotechnology) and p35 (1 : 150 dilution, sc-7925, Santa Cruz Biotechnology) at 4��C overnight. Labelling was detected by adding biotinylated secondary antibodies (Maxim-Bio, Fuzhou, China), avidin�Cbiotin complex (Maxim-Bio), and stained with DAB (Maxim-Bio). Finally, sections were counterstained with haematoxylin and viewed under a R428 light microscope. A negative control was obtained by replacing the primary antibody with a normal murine IgG, and human colorectal cancer tissue was used as positive control. Expressions of EBI3 and p35 were scored according to the positive percentage and staining intensity of the E-64 stained cancer cells. The percentage positivity was scored as 0 (0�C25%), 1 (26�C50%), 2 (51�C75%), and 3 (> 75%). The staining intensity was scored as 0 (no staining), 1 (weakly stained), 2 (moderately stained), and 3 (strongly stained). If the product of multiplication between staining intensity and the percentage of positive cells was �� 2, it was taken as immunoreaction positive (+). Statistical analysis All statistical analyses were carried out using SPSS13.0 software. Fisher's exact test was used to analyse the correlation between the clinicopathological characteristics and EBI3, p35 expression. Survival curves were obtained with the Kaplan-Meier method. Multivariate analysis was performed using Cox proportional hazards regression. p selleck chemical immunoreactivity was present in 41 cases (51.3%, 41/80). Compared with no staining in the normal nasopharyngeal mucosa, EBI3 and p35 were over-expressed in the cytoplasm of the nasopharyngeal carcinoma cells (Fig. 1). Fig. 1 EBI3 and p35 expression in nasopharyngeal carcinoma samples by immunohistochemistry. A, E) No staining of EBI3 and p35 in non-tumoural nasopharyngeal mucosa (��200). B, C, D) Positive expression of EBI3 in the cytoplasmic of nasopharyngeal carcinoma ... Correlation of EBI3 and p35 expression with clinicopathological factors of nasopharyngeal carcinoma patients To further investigate the clinical significance of IL-35 in nasopharyngeal carcinoma specimens, the correlation between IL-35 expression and clinicopathological factors was explored. As summarised in Table 1, EBI3 expression was significantly associated with tumour stage (p = 0.003) and lymph node metastasis (p = 0.018).