Ab9373

Human gallbladder bile was obtained from 54 CCA patients in O. viverrini endemic areas who had undergone hepatectomy. Bile samples were collected from Srinagarind Hospital, Khon Kaen University, Khon Kaen, Thailand. Patient profiles, clinical information, and tumor classification were also obtained. Five CCA tissues and 5 non-CCA adjacent normal tissues were included as positive and negative controls. Western blot analysis was performed as previously described. 17 Briefly, 5 µL of bile sample was separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with 20 mA for 2 h. Separated proteins were then transferred to nitrocellulose membrane. Non-specific binding was blocked by incubating membrane in 5% skimmed milk in PBS with 0.05% Tween 20 (PBST) at 25°C for 1 h. The membrane was then incubated for 1 h with 1:1000 diluted rabbit anti-alpha 1 antitrypsin antibody (polyclonal, ab9373; Abcam) in 2% skimmed milk in PBST at 25°C. After washing with PBST, the membrane was incubated in diluted 1:1000 anti-rabbit HRP-conjugated ab97051 (Abcam) in 2% skimmed milk in PBST at 25°C for 1 h. The membrane was then washed with PBST and PBS, and reactive band was visualized with 3,3′-diaminobenzidine tetrahydrochloride solutions (DAB; Sigma Chemical, USA).

A CCA tissue microarray (TMA, n = 354 and control, n = 2) was constructed by the Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand. IHC reactions were performed on 4-µm-thick paraffin sections of CCA TMA on silane-coated slides (Sigma Chemical, USA) using the immunoperoxidase method. 16 The paraffin sections were de-waxed in xylene and rehydrated in serial dilution of ethanol. Antigen retrieval was performed in citrate buffer pH 6.0 under high pressure. Endogenous peroxidase activity was blocked in methanol with 3% hydrogen peroxide (HO). Non-specific binding was blocked by incubation in 5% normal horse serum, followed by overnight incubation at 4°C with 1:300 diluted rabbit anti-alpha 1 antitrypsin antibody (polyclonal, ab9373; Abcam, USA). Phosphate-buffered saline (PBS) was used instead of primary antibody in the control sections. Sections were washed with PBS and incubated with the specific secondary antibodies for 30 min at room temperature (1:300 diluted anti-rabbit horseradish peroxidase (HRP) conjugated, ab97051; Abcam). Slides were subsequently counterstained with Mayer's™ hematoxylin, dehydrated, cleaned, and mounted. All tissue slides were histopathologically evaluated by M.L., B.S., and the senior gastrointestinal pathologist, C.P.