Protocol for mapping tumor infiltration by murine T cells using multiplex immunofluorescence

Here, we present a 7-plex immunofluorescence staining protocol that enables the spatial analysis of murine tumor tissues to investigate the complex interplay between immune cells and the tumor microenvironment. We used a sequential antibody labeling strategy that identifies CD4⁺ and CD8⁺ T cells and characterizes their activation and regulatory states. This protocol also maps T cell infiltration within tumors, allowing researchers to investigate immune cell distribution, interactions, and the dynamic biology of the tumor immune landscape in murine tumor models. For complete details on the use and execution of this protocol, please refer to Lim et al.¹.