Il 12rβ2

Tissue microarrays were constructed according to a previous research report.25 Two different tissue microarray blocks extracted from each case (3‐mm‐diameter each) were used for TMA construction.
Monoclonal rabbit antihuman antibodies P35, EBI3, gp130 and IL‐12Rβ2 (1:100 dilution) were purchased from Abcam (Cambridge, UK). IHC was used to detect the expression of P35, EBI3, gp130 and IL‐12Rβ2 in tissues of ICC, and surgical produces were carried out as described.26 Two experienced observers were responsible for sliced evaluation in a double blind method. Expression of IL‐35, EBI3 gp130 and IL‐12Rβ2 was evaluated by a semiquantitative scoring system.27 The final score was made by positively stained tumor cells (0, no positive tumor cells; 1, <10%; 2, 10%‐35%; 3, 35%‐75%; 4, >75%) multiplied by staining intensity (1, no staining; 2, weak; 3, moderate; 4, strong). Eventually, total scores ≥8 were defined as high expression and those <8 were defined as low expression. Representative images of positive and negative controls for IHC are shown in Figure S1.

Protein (30 μg) was separated by 10% SDS‐PAGE electrophoresis, then transferred to PVDF (Millipore, Temecula, CA, USA), and the membranes were blocked in 5% BSA for 1 hour, then incubated with primary antibodies (P35, EBI3, gp130 and IL‐12Rβ2 (1:1000 dilution; Abcam) at 4°C overnight. β‐Actin (Kangcheng, Shanghai, China) was used as an internal control. After 12 hours, the membranes were incubated with secondary antibody (Kangcheng) for 1 hour at room temperature. Finally, the protein bands were detected by enhanced chemiluminescent (ECL) substrate and processed by Image, Lab software (Bio‐Rad, Mississauga, ON, Canada).