Protocol for iterative indirect immunofluorescence imaging in cultured cells, tissue sections, and metaphase chromosome spreads
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 met...
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| Published in | STAR protocols Vol. 5; no. 3; p. 103190 |
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| Main Authors | , , , , |
| Format | Journal Article |
| Language | English |
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United States
Elsevier Inc
20.09.2024
Elsevier |
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| Online Access | Get full text |
| ISSN | 2666-1667 2666-1667 |
| DOI | 10.1016/j.xpro.2024.103190 |
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| Abstract | 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
[Display omitted]
•Three variants of an iterative imaging protocol for cellular and subcellular analysis•Highly multiplexed immunofluorescence staining and antibody elution in diverse samples•Streamlined image processing steps for cultured cells, tissue sections, and chromosomes
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
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. |
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| AbstractList | 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.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. 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 •Three variants of an iterative imaging protocol for cellular and subcellular analysis•Highly multiplexed immunofluorescence staining and antibody elution in diverse samples•Streamlined image processing steps for cultured cells, tissue sections, and chromosomes Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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. 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 [Display omitted] •Three variants of an iterative imaging protocol for cellular and subcellular analysis•Highly multiplexed immunofluorescence staining and antibody elution in diverse samples•Streamlined image processing steps for cultured cells, tissue sections, and chromosomes Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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. 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. . 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. |
| ArticleNumber | 103190 |
| Author | Janes, Kevin A. Fallahi-Sichani, Mohammad Hsu, Jeffrey Nguyen, Kimberly T. Bujnowska, Magda |
| Author_xml | – sequence: 1 givenname: Jeffrey surname: Hsu fullname: Hsu, Jeffrey organization: Medical Scientist Training Program, University of Virginia, Charlottesville, VA 22908, USA – sequence: 2 givenname: Kimberly T. surname: Nguyen fullname: Nguyen, Kimberly T. organization: Medical Scientist Training Program, University of Virginia, Charlottesville, VA 22908, USA – sequence: 3 givenname: Magda surname: Bujnowska fullname: Bujnowska, Magda organization: Medical Scientist Training Program, University of Virginia, Charlottesville, VA 22908, USA – sequence: 4 givenname: Kevin A. surname: Janes fullname: Janes, Kevin A. organization: Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA – sequence: 5 givenname: Mohammad orcidid: 0000-0003-0917-3525 surname: Fallahi-Sichani fullname: Fallahi-Sichani, Mohammad email: fallahi@virginia.edu organization: Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA |
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| Cites_doi | 10.1186/gb-2006-7-10-r100 10.7554/eLife.31657 10.1038/ncomms14836 10.1038/ncomms9390 10.1038/s41592-021-01308-y 10.1016/j.celrep.2022.111147 10.1126/science.aar7042 10.1093/bioinformatics/btac544 10.1038/nmeth.2019 10.1023/B:VISI.0000029664.99615.94 10.21769/BioProtoc.4712 |
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| SubjectTerms | Animals Cancer Cell Biology Cells, Cultured Fluorescent Antibody Technique, Indirect - methods Humans Metaphase Microscopy Microscopy, Fluorescence - methods Protocol Systems biology |
| Title | Protocol for iterative indirect immunofluorescence imaging in cultured cells, tissue sections, and metaphase chromosome spreads |
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