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Related Concept Videos

Immunofluorescence Microscopy01:12

Immunofluorescence Microscopy

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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: Aug 17, 2025

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging
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Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

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Multiplex Spatial Protein Detection by Combining Immunofluorescence with Immunohistochemistry.

Wenfei Kang1, Anthony Santella2, Eric Rosiek2

  • 1Molecular Cytology Core Facility, Memorial Sloan Kettering Cancer Center, New York, NY, USA. kangw@mskcc.org.

Methods in Molecular Biology (Clifton, N.J.)
|December 13, 2022
PubMed
Summary

This study introduces a flexible method combining multiplexed immunofluorescence (IF) and sequential immunohistochemistry (IHC) for detecting 10 antigens in one tissue section. This approach enhances spatial protein analysis without expensive reagents or limited antibody panels.

Keywords:
AEC chromogenAlignmentImmunofluorescence (IF)Immunohistochemistry (IHC)Leica Bond RXMultiplexVisualization

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A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections
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Area of Science:

  • Biotechnology
  • Histology
  • Immunohistochemistry

Background:

  • Multiplexed staining technologies are advancing for spatial protein relationship analysis at cellular resolution.
  • Current methods often face limitations in antigen detection capacity, cost, and flexibility with antibody panels.

Purpose of the Study:

  • To describe a novel, flexible method for multiplexed staining of paraffin-embedded tissue sections.
  • To enable detection of multiple antigens in a single section using readily available equipment and reagents.

Main Methods:

  • Combined multiplexed immunofluorescence (IF) with sequential immunohistochemistry (IHC) using AEC chromogen on Leica Bond processors.
  • Developed an image analysis workflow with nonlinear warping for aligning multiple staining passes at the single-cell level.

Main Results:

  • Successfully detected 10 antigens in a single paraffin-embedded tissue section while preserving tissue integrity.
  • Demonstrated flexibility for using custom antibody combinations, not limited to pre-validated panels.

Conclusions:

  • The described method offers a cost-effective and flexible approach for multiplexed protein detection in tissues.
  • This technique facilitates high-resolution spatial analysis of multiple proteins, advancing biomarker discovery and research.