The study protocol conformed to the ethical guidelines of the Declaration of Helsinki (1975). All patients provided written

informed consent for the analysis of the biopsy specimens or drainage bile. The protocol for this study was approved by the ethical committee of Kanazania University, Tokyo Women’s Medical University and University of Tsukuba. Differential glycan profiling of tissue sections was performed essentially as described.21 Briefly, formalin-fixed, paraffin-embedded ICC tissue sections were deparaffinized, and the relevant tissue fragments including cancerous (n = 45) and normal bile duct epithelia (BDE) (n = 38) lesions (corresponding to 1.0 mm square and 5 μm thickness, respectively) were then scratched www.selleckchem.com/products/erastin.html from the glass slide using a needle (gauge size: 21 G) under a microscope. Total protein extracts from the scratched tissue fragments thus obtained were fluorescence-labeled with 10 μg of Cy3-succimidyl ester (SE; Amersham selleck chemical Biosciences, Tokyo, Japan). After blocking free Cy3-SE with 0.5 M glycine in Tris-buffered saline containing 1% Triton X-100 (TBSTx), an aliquot (¼) was applied to a lectin microarray slide. Fluorescence

signals were measured on a GlycoStation scanner (Moritex Co., Tokyo, Japan). The obtained lectin microarray data were analyzed on the basis of normalized signal intensities as described,23 where the lectin showing the strongest signal intensity (max intensity) was assigned a value of 1.0. The values are presented as the median ± standard error of the mean (SEM). A two-sided Welch or Student t test was used to compare the clinicopathological data between groups. All calculations were performed using Origin version 7.5 software

for Windows (OriginLab Co., Northampton, MA). Receiver operating characteristic (ROC) curve analysis was performed to evaluate the differences between ICC and benign disease on the bases of sensitivity and specificity at various cutoff levels. An area under the ROC curve (AUC) of 1.0 indicates perfect discrimination, whereas an area of GNA12 0.5 indicates that the test discriminates no better than chance.24 WFA staining was performed using biotinylated WFA (Vector Co., Burlingame, UK). Detection was made with Histofine Simple Stain MAX-PO (Nichirei Co., Tokyo, Japan). The tissue sections were deparaffinized and then autoclaved to enhance the WFA reactivity. After cooling to room temperature, endogenous peroxidase was blocked by incubating the sections in methanol containing 0.3% hydrogen peroxide. The tissue sections were blocked with phosphate-buffered saline (PBS) containing 1% (wt/vol) bovine serum albumin (BSA), and the sections were incubated with 2 μg/mL of biotinylated WFA in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid for 1 hour at room temperature. The sections were incubated with streptavidin–peroxidase reagents, reacted with 3,3′-diaminobenzidine tetrahydrochloride for visualization, and counterstained with hematoxylin.