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Protocol for investigating tertiary lymphoid structures in human and murine fixed tissue sections using Opal™-TSA
Luke T Quigley1, Lokman Pang1, Elnaz Tavancheh1
1Olivia Newton-John Cancer Research Institute, and School of Cancer Medicine, La Trobe University, Heidelberg, VIC 3084, Australia.
STAR Protocols
|January 12, 2023
Summary
A new protocol allows researchers to identify and characterize tertiary lymphoid structures (TLSs) in tissues. This method uses multiplex immunohistochemistry to visualize up to seven markers, aiding in the study of tumor-associated immune responses.
Area of Science:
- Immunology
- Pathology
- Cancer Research
Background:
- Tertiary lymphoid structures (TLSs) are immune cell aggregates found in non-lymphoid tissues, often associated with disease.
- Their presence in tumors can influence patient prognosis and response to therapy.
- Current methods for identifying and characterizing TLSs in situ are limited.
Purpose of the Study:
- To develop and validate a robust protocol for the in situ identification and characterization of TLSs.
- To enable simultaneous detection of multiple markers within TLSs.
- To establish a grading system for assessing TLS maturity.
Main Methods:
- Multiplex immunohistochemistry (IHC) using Opal™-tyramide signal amplification on human and murine tissue sections.
- Simultaneous detection of up to seven markers, including six antigens and a DAPI counterstain.
- Development of a grading system to differentiate between immature and mature TLSs.
Main Results:
- The protocol successfully enables in situ interrogation and characterization of TLSs.
- Simultaneous detection of multiple markers provides detailed insights into TLS composition.
- The grading system effectively distinguishes between immature and mature TLS phenotypes.
Conclusions:
- This protocol offers a powerful tool for studying TLSs in various pathological contexts, particularly in cancer.
- Accurate identification and characterization of TLSs can advance our understanding of immune responses in tumors.
- The developed grading system facilitates standardized assessment of TLS maturation.
