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Immunofluorescence Microscopy

Immunofluorescence Microscopy

A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) and fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI), and acridine orange.

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Related Experiment Video

Updated: Jul 2, 2025

Author Spotlight: Efficient Detection of Immune Cell-Infiltration in Cancer Tissues Using Fluorescent Immunohistochemistry

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.

Journal of Visualized Experiments : Jove

|February 19, 2024

Summary

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.

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Last Updated: Jul 2, 2025

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Published on: January 26, 2024

Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry

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Published on: July 26, 2019

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Published on: January 9, 2019

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.