Protocol for iterative indirect immunofluorescence imaging in cultured cells, tissue sections, and metaphase chromosome spreads

Jeffrey Hsu; Kimberly T. Nguyen; Magda Bujnowska; Kevin A. Janes; Mohammad Fallahi-Sichani

STAR Protocols (Sep 2024)

Protocol for iterative indirect immunofluorescence imaging in cultured cells, tissue sections, and metaphase chromosome spreads

Jeffrey Hsu,
Kimberly T. Nguyen,
Magda Bujnowska,
Kevin A. Janes,
Mohammad Fallahi-Sichani

Affiliations

Jeffrey Hsu: Medical Scientist Training Program, University of Virginia, Charlottesville, VA 22908, USA; Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA
Kimberly T. Nguyen: Medical Scientist Training Program, University of Virginia, Charlottesville, VA 22908, USA; Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA
Magda Bujnowska: Medical Scientist Training Program, University of Virginia, Charlottesville, VA 22908, USA; Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA
Kevin A. Janes: Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA; Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA; UVA Comprehensive Cancer Center, University of Virginia, Charlottesville, VA 22908, USA
Mohammad Fallahi-Sichani: Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA; UVA Comprehensive Cancer Center, University of Virginia, Charlottesville, VA 22908, USA; Corresponding author

Journal volume & issue: Vol. 5, no. 3
p. 103190

Abstract

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Summary: We present a protocol to generate highly multiplexed spatial data at cellular and subcellular resolutions using iterative indirect immunofluorescence imaging (4i). We describe streamlined steps for using 4i across fixed cultured cells, formalin-fixed paraffin-embedded (FFPE) tissue sections, and metaphase chromosome spreads. We detail procedures for sample preparation, antibody and DNA staining, immunofluorescence imaging, antibody elution, and image processing. This protocol is adapted for high-throughput analysis of fixed cultured cells and addresses sample-specific challenges such as intrinsic tissue autofluorescence and chromosome fragility.For complete details on the use and execution of this protocol for fixed cultured cells, please refer to Comandante-Lou et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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Abstract

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