Immunohistochemical Staining of Cell Adhesion Proteins

Antigens were retrieved by heat-induced epitope retrieval in 0.01 M citrate buffer with 0.05% Tween 20, pH 6 for N-cadherin and β-catenin and in Target Retrieval Solution, pH 9 (Dako Australia Pty Ltd., New South Wales, Australia) for EPHA2 labeling, as described elsewhere.⁵¹ (link) Sections were blocked with 3% goat or donkey serum (Sigma-Aldrich Pty. Ltd., Sydney, Australia), incubated with mouse anti-N-cadherin (1:200; Life Technologies Australia Pty. Ltd., Victoria, Australia) or mouse anti-β-catenin (1:200; BD Transduction Laboratories, San Diego, CA, USA) or goat anti-mouse (m) EPHA2 (1:40; R&D Systems, Inc., Minneapolis, MN, USA) primary antibody followed by goat anti-mouse or donkey anti-goat IgG Alexa Fluor 488-conjugated (1:1000; Life Technologies Australia Pty. Ltd.) secondary antibody; control sections were incubated with equivalent amount of mouse or goat IgG. Labeled sections were mounted in Prolong AntiFade with DAPI (Life Technologies Australia Pty. Ltd.) and imaged as previously described.⁵² (link)