UltraPlex Hapten-Based Multiplexed Fluorescent Immunohistochemistry

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 2350; p. 267
• Main Authors: Levin, Matt, Flor, Amy C, Snyder, Helen, Kron, Stephen J, Schwartz, David
• Format: Journal Article
• Language: English
• Published: United States 2021
• Subjects: Biomarkers; Biomarkers, Tumor; Data Analysis; Fluorescent Antibody Technique; Haptens; Humans; Image Processing, Computer-Assisted - methods; Immunohistochemistry - methods; Neoplasms - metabolism; Neoplasms - pathology; Staining and Labeling; Immunohistochemistry; Microscopy; Immunofluorescence; Multiplex imaging; Antibody; Hapten
• ISSN: 1940-6029
• DOI: 10.1007/978-1-0716-1593-5_17

The UltraPlex method for multiplexed two-dimensional fluorescent immunohistochemistry is described, in which hapten tags conjugated to primary antibodies facilitate multiplexed imaging of four or more antigens per tissue section at once. Anti-hapten secondary antibodies labeled with fluorophores provide amplified signal for detection, which is accomplished using a standard fluorescent microscope or digital slide scanner. The protocol is rapid and straightforward and utilizes conventionally prepared tissue samples. The resulting staining is highly sensitive and specific, enabling high-resolution imaging of multiple cellular subtypes within tissue samples. Tumor cells and tumor-infiltrating lymphocytes are presented as examples. Multiple 4-plex-stained tissue samples can be digitally overlaid to create 8-plex (or more) high-content images, enabling visualization of distribution of complex cellular subtypes across tissues.