Ki-67 Membranous Staining: Biologically Relevant or an Artifact of Multiplexed Immunofluorescent Staining.

作者信息

Wang Dan, Pang Zhengyu, Clarke Gina M, Nofech-Mozes Sharon, Liu Kela, Cheung Alison M Y, Filkins Robert J, Yaffe Martin J

机构信息

*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.

出版信息

Appl Immunohistochem Mol Morphol. 2016 Jul;24(6):447-52. doi: 10.1097/PAI.0000000000000202.

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.