Protocol for visualization and quantitative analysis of murine lung immunological architecture using iBALT-UNMAP following bacterial challenge.

The spatial distribution of adaptive immune cells is a critical determinant of the quality and magnitude of mucosal immunity. Here, we present a protocol for tyramide signal amplification (TSA) immunofluorescence that preserves the lung architecture and quantifies inducible bronchus-associated lymphoid tissue (iBALT) following murine Bordetella bronchiseptica infection. Starting from paraffin-embedded lungs, we describe steps for staining, imaging, and morphometric analysis. This protocol can be optimized for other pathogens and experimental settings to allow mapping of spatial immune responses within the respiratory mucosa. For complete details on the use and execution of this protocol, please refer to First et al.1.