Richard allan scientific cytoseal xyl

Paraffin sections were immunostained by Dako Autostainer using Dako Envision™ FLEX+ system (K8012; Dako, Glostrup, Denmark) as described in our previous study (15 (link)). Briefly, the sections were deparaffinized, epitopes were unmasked in PT-link with low pH target retrieval solution (Dako) and then blocked with peroxidase blocking solution (Dako) for 5 min. The slides were incubated at room temperature with polyclonal rabbit anti-human ZEB1 antibody (cat. no. HPA027524; 1:500; Sigma-Aldrich, St. Louis, USA), followed by incubation with rabbit linker for 15 min and horseradish peroxidase for 30 min at room temperature. The slides were subsequently stained with 3,3′-diaminobenzidine tetrahydrochloride for 10 min and counter-stained with hematoxylin, dehydrated, and mounted in Richard-Allan Scientific Cytoseal XYL (Thermo Fisher Scientific, Waltham, MA, USA). A known ZEB1-positive human esophageal carcinoma slide was used as positive control. Serial negative controls were tested by the same concentration of normal rabbit serum as a substitute for the rabbit anti-human ZEB1 antibody.