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Updated: Jul 2, 2025
Author Spotlight: Efficient Detection of Immune Cell-Infiltration in Cancer Tissues Using Fluorescent Immunohistochemistry
Published on: January 26, 2024
Multiplex Cyclic Fluorescent Immunohistochemistry
Yun Chen1, Hui Zhang2, Yaodong Fan3
1Department of Pathology, The Third Affiliated Hospital of Kunming Medical University & Yunnan Cancer Hospital.
Insights
Multiplex cyclic fluorescence immunohistochemistry enables simultaneous detection of multiple markers in tissue samples. This reproducible technique aids in analyzing immune cell subsets for immunotherapy research and clinical applications.
Area of Science:
- Immunology
- Pathology
- Biotechnology
Background:
- The tumor microenvironment (TME) comprises complex interactions crucial for disease progression and treatment response.
- Characterizing immune cell subsets and protein expression within the TME is vital for prognostic and therapeutic strategies.
- Multiplexed immunohistochemistry techniques have emerged to address the need for simultaneous marker detection.
Purpose of the Study:
- To describe a workflow for multiplex cyclic fluorescence immunohistochemistry (mcf-IHC).
- To demonstrate the application of mcf-IHC in quantifying lymphocyte subsets.
- To highlight the value of mcf-IHC for immunotherapy research and clinical applications.
Main Methods:
- A workflow for mcf-IHC was developed, utilizing formalin-fixed paraffin-embedded (FFPE) tissue slides.
- The protocol involves antigen retrieval, cyclic antibody incubation, and staining, similar to standard immunohistochemistry.
- Optimization of antigen retrieval and antibody concentrations was performed to enhance the signal-to-noise ratio.
Main Results:
- The study successfully established and validated a reproducible mcf-IHC workflow.
- The assay allows for the simultaneous detection and quantification of multiple markers, including lymphocyte subsets.
- Optimized conditions improved the signal-to-noise ratio for accurate analysis.
Conclusions:
- Multiplex cyclic fluorescence immunohistochemistry is a valuable and reproducible tool for detailed TME analysis.
- This technique facilitates a comprehensive understanding of cell function and intercellular interactions.
- mcf-IHC holds significant potential for advancing immunotherapy research and clinical diagnostics.
Abstract:
The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and spatially organizing immune cell subsets and target proteins are crucial for prognostic and therapeutic purposes. This has led to the development of multiplexed immunohistochemistry staining methods. Multiplex fluorescence immunohistochemistry allows the simultaneous detection of multiple markers, facilitating a comprehensive understanding of cell function and intercellular interactions. In this paper, we describe a workflow for the multiplex cyclic fluorescent immunohistochemistry assay and its application in the quantification analysis of lymphocyte subsets. The multiplex cyclic fluorescent immunohistochemistry staining follows similar steps and reagents as standard immunohistochemistry, involving antigen retrieval, cyclic antibody incubation, and staining on a formalin-fixed paraffin-embedded (FFPE) tissue slide. During the antigen-antibody reaction, a mixture of antibodies from different species is prepared. Conditions, such as antigen retrieval time and antibody concentration, are optimized and validated to increase the signal-to-noise ratio. This technique is reproducible and serves as a valuable tool for immunotherapy research and clinical applications.
