3D Multiplexed Immunohistochemistry Using Opal-TSA Amplification for Enhanced Imaging of Pulmonary TB Lesions

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Summary Mycobacterium tuberculosis hijacks the host immune system and persists for several years before the onset of active disease. Spatial characterization of epithelial compartments, immune cell populations and bacteria simultaneously within tissue specimens provide significant information about host pathogen interactions. Here, we present a protocol to detect multiple protein markers using Opal™-TSA conjugated fluorescent dyes in free floating 10% neutral buffered formalin fixed thick tissue sections (50-100 μm), with minimal additional tissue processing not requiring specialized equipment. Use of thick sections provides more information as compared to classic thin microtomy sections (3-10 μm), including the capacity for Z stacking and three-dimensional image rendering. Importantly, reduced tissue processing of samples with this method preserves endogenous fluorescent reporter signal. Use of Opal™-TSA conjugated fluorescent dyes enhances the sensitivity of low expressing proteins and supports the use of primary antibodies raised in the same species. Before you begin This protocol describes the specific use of Mtb infected mouse lungs, but is applicable to any tissue type and species of origin. Institutional permissions Obtain institutional permission to perform animal studies and collect tissues under an approved Institutional Animal Care and Use Committee (IACUC) or Institutional Review Board protocol. Our protocol was approved by Boston University’s Institutional Animal Care and Use Committee (IACUC protocol number PROTO201800218). Mice B6J.C3- Sst1 C3HeB/Fej Krmn and B6. Sst1S, ifnb-YFP mice were developed in our laboratory (available from MMRRC stock # 043908-UNC). Highlights Compatible with Opal™-TSA conjugated fluorescent dyes resulting in enhanced signal sensitivity with low background noise Reduced tissue processing preserves endogenous fluorescent reporter signals Compatible with primary antibodies raised in the same species Doesn’t require specialized automated instruments Graphical Abstract