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

Abstract

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 m), 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 m), 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 beginThis protocol describes the specific use of Mtb infected mouse lungs, but is applicable to any tissue type and species of origin. Institutional permissionsObtain 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 Universitys Institutional Animal Care and Use Committee (IACUC protocol number PROTO201800218). MiceB6J.C3-Sst1C3HeB/Fej Krmn and B6. Sst1S, ifnb-YFP mice were developed in our laboratory (available from MMRRC stock # 043908-UNC). HighlightsO_LICompatible with Opal-TSA conjugated fluorescent dyes resulting in enhanced signal sensitivity with low background noise C_LIO_LIReduced tissue processing preserves endogenous fluorescent reporter signals C_LIO_LICompatible with primary antibodies raised in the same species C_LIO_LIDoesnt require specialized automated instruments C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/619885v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@3e3439org.highwire.dtl.DTLVardef@1d6ba85org.highwire.dtl.DTLVardef@6f9595org.highwire.dtl.DTLVardef@1c573ae_HPS_FORMAT_FIGEXP M_FIG C_FIG