Protocol to study immunodynamics in the tumor microenvironment using a tyramide signal amplification-based immunofluorescent multiplex panel

Immunodynamics in the tumor microenvironment can be precisely examined by using multiple antigen identification approaches. Here, we present a protocol for capturing expression levels of multiple target proteins in the same specimen at single-cell resolution using a tyramide signal amplification-based immunofluorescent multiplexing system. We describe steps for tumor tissue microarray preparation, multiplex immunohistochemistry staining, image acquisition, and quantification. This protocol can quantify immune cells in tissues from patients or experimental disease models at a protein level. For complete details on the use and execution of this protocol, please refer to Chung et al. (2023),1 Tang et al. (2022),2 and Tang et al. (2022).3 [Display omitted] •Low-background multiplex immunohistochemistry staining in FFPE samples•Image acquisition for detecting multiple protein expressions in tumor microarray•Unbiased quantification of antigens’ colocalization using inForm software Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Immunodynamics in the tumor microenvironment can be precisely examined by using multiple antigen identification approaches. Here, we present a protocol for capturing expression levels of multiple target proteins in the same specimen at single-cell resolution using a tyramide signal amplification-based immunofluorescent multiplexing system. We describe steps for tumor tissue microarray preparation, multiplex immunohistochemistry staining, image acquisition, and quantification. This protocol can quantify immune cells in tissues from patients or experimental disease models at a protein level.