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 • Understanding lung immunological architecture is critical for interpreting immune responses • We established a tool to unify the nomenclature of lung B/T cell aggregates • Unbiased bioinformatic approaches to compare iBALT and aggregates across challenges • Defining lung immune architecture reveals unique host responses to specific pathogens Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.
First et al. (Mon,) studied this question.