title: Ki-67 Membranous Staining: Biologically Relevant or an Artifact of Multiplexed Immunofluorescent Staining.
abstract: In the process of developing a multiplex of 8 common breast cancer biomarkers (Her2/neu, estrogen receptor, progesterone receptor, Ki-67, aldehyde dehydrogenase-1, NaK-ATPase, cytokeratin 8/18, and myosin smooth muscle) on a single formalin-fixed paraffin-embedded slide using a sequential staining, imaging, and dye bleaching technology developed by General Electric Company, membranous Ki-67 staining was observed and colocalized with Her2/neu staining. Using immunohistochemistry as gold standards, we discovered that membranous Ki-67 was an artifact caused by the binding of cyanine 5-conjugated rabbit polyclonal Ki-67 antibody to a secondary cyanine 3-conjugated donkey anti-rabbit antibody which was previously applied and bound to rabbit Her2/neu antibody in our multiplexing experiment. After blocking with rabbit serum, a successful protocol for 8 biomarker multiplexing without cross-reactivity of antibodies from the same species was developed.
authors.affiliation: *Physical Sciences, Sunnybrook Research Institute §Department of Anatomic Pathology, Sunnybrook Health Sciences Centre Departments of ‡Laboratory Medicine and Pathobiology ∥Departments of Medical Biophysics and Medical Imaging, Faculty of Medicine, University of Toronto, Toronto, ON, Canada †Diagnostics and Biomedical Technologies, Global Research Center, General Electric Company, Niskayuna, NY.
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.forename: Zhengyu
authors.initials: Z
authors.lastname: Pang
authors.forename: Gina M
authors.initials: GM
authors.lastname: Clarke
authors.forename: Sharon
authors.initials: S
authors.lastname: Nofech-Mozes
authors.forename: Kela
authors.initials: K
authors.lastname: Liu
authors.forename: Alison M Y
authors.initials: AM
authors.lastname: Cheung
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Filkins
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Yaffe
mesh_terms: D016477:Artifacts; D001943:Breast Neoplasms / Q000378:metabolism; D004396:Coloring Agents; D005455:Fluorescent Antibody Technique; D006801:Humans; D019394:Ki-67 Antigen / Q000378:metabolism*
chemical_list: D004396:Coloring Agents; D019394:Ki-67 Antigen