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Updated: Apr 5, 2026
Author Spotlight: Efficient Detection of Immune Cell-Infiltration in Cancer Tissues Using Fluorescent Immunohistochemistry
Published on: January 26, 2024
Ki-67 Membranous Staining: Biologically Relevant or an Artifact of Multiplexed Immunofluorescent Staining
Dan Wang1, Zhengyu Pang, Gina M Clarke
1*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.
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.
