Protocol for 3D multiplexed fluorescent imaging of pulmonary TB lesions using Opal-TSA dyes for signal amplification

To detect multiple protein markers, here we present a protocol for imaging pulmonary tuberculosis (PTB) lesions from murine lungs using Opal-tyramide signal amplification (TSA) conjugated fluorescent dyes in free-floating formalin-fixed thick lung sections (50-100 μm). We describe steps for preparing tissue sections, permeabilization and antigen retrieval, and multiplexing with antibodies raised in the same species. This protocol has been optimized to preserve tissue integrity and endogenously expressed fluorescent reporter signals and nuclear staining. It enhances the signal-to-background ratio and enables 3D image rendering.