Tertiary lymphoid structures (TLSs) are ectopic lymphoid aggregates that can develop within or adjacent to tumors, but protocols that can accurately identify and characterize TLSs are lacking. Here, we present a protocol for the in situ interrogation and characterization of TLSs in human and murine tissue sections using Opal™-tyramide signal amplification multiplex immunohistochemistry. This protocol enables simultaneous detection of up to 7 markers (6 antigens and a DAPI counterstain). We also describe a grading system to identify immature and mature TLSs.
Funding
We acknowledge The Ian Potter Foundation for providing funds to purchase the Vectra System and BondRX. This project was partly funded by the National Medical Health and Research Council (NHMRC) of Australia (GNT1125951) , by the Austin Medical Research Foundation, and by Tour de Cure. L.P. is supported by the La Trobe University Full-Fee Research Scholarship and the La Trobe University Postgraduate Research Scholarship. J.D.G.D. was supported by Cure Cancer Australia through the Cancer Australia Priority-Driven Cancer Research Scheme (#1187815). A.B. is the recipient of a Fellowship from the Victorian Government Department of Health and Human Services acting through the Victorian Cancer Agency.