Formalin-fixed, paraffin-embedded sections from the above-described 685 tumors were analyzed for claudin-10 protein expression using the Dako EnVision Flex + System (K8012; Dako, Glostrup, Denmark). The claudin-10 antibody was a rabbit polyclonal antibody purchased from Invitrogen (cat # 38–8400; Waltham, MA), applied at a 1:100 dilution following antigen retrieval in LpH buffer (pH 6.0).
Following deparaffinization, sections were treated with EnVision™ Flex + mouse linker (15 min) and EnVision™ Flex/HRP enzyme (30 min) and stained for 10 min with 3′3-diaminobenzidine tetrahydrochloride (DAB), counterstained with hematoxylin, dehydrated and mounted in Toluene-Free Mounting Medium (Dako). Positive control consisted of normal pancreas. In negative controls, the primary antibody was replaced with rabbit serum diluted to the same concentration as the primary antibody.
Following deparaffinization, sections were treated with EnVision™ Flex + mouse linker (15 min) and EnVision™ Flex/HRP enzyme (30 min) and stained for 10 min with 3′3-diaminobenzidine tetrahydrochloride (DAB), counterstained with hematoxylin, dehydrated and mounted in Toluene-Free Mounting Medium (Dako). Positive control consisted of normal pancreas. In negative controls, the primary antibody was replaced with rabbit serum diluted to the same concentration as the primary antibody.