Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics
Abstract Tissues are complex environments where different cell types are in constant interaction with each other and with non-cellular components. Preserving the spatial context during proteomics analyses of tissue samples has become an important objective for different applications, one of the most...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-10-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-43435-w |
| _version_ | 1827634285155188736 |
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| author | François Rivest Deniz Eroglu Benjamin Pelz Joanna Kowal Alexandre Kehren Vytautas Navikas Maria Giuseppina Procopio Pino Bordignon Emilie Pérès Marco Ammann Emmanuel Dorel Sylvain Scalmazzi Lorenzo Bruno Matthieu Ruegg Gabriel Campargue Gilles Casqueiro Lionel Arn Jérôme Fischer Saska Brajkovic Pierre Joris Marco Cassano Diego Dupouy |
| author_facet | François Rivest Deniz Eroglu Benjamin Pelz Joanna Kowal Alexandre Kehren Vytautas Navikas Maria Giuseppina Procopio Pino Bordignon Emilie Pérès Marco Ammann Emmanuel Dorel Sylvain Scalmazzi Lorenzo Bruno Matthieu Ruegg Gabriel Campargue Gilles Casqueiro Lionel Arn Jérôme Fischer Saska Brajkovic Pierre Joris Marco Cassano Diego Dupouy |
| author_sort | François Rivest |
| collection | DOAJ |
| description | Abstract Tissues are complex environments where different cell types are in constant interaction with each other and with non-cellular components. Preserving the spatial context during proteomics analyses of tissue samples has become an important objective for different applications, one of the most important being the investigation of the tumor microenvironment. Here, we describe a multiplexed protein biomarker detection method on the COMET instrument, coined sequential ImmunoFluorescence (seqIF). The fully automated method uses successive applications of antibody incubation and elution, and in-situ imaging enabled by an integrated microscope and a microfluidic chip that provides optimized optical access to the sample. We show seqIF data on different sample types such as tumor and healthy tissue, including 40-plex on a single tissue section that is obtained in less than 24 h, using off-the-shelf antibodies. We also present extensive characterization of the developed method, including elution efficiency, epitope stability, repeatability and reproducibility, signal uniformity, and dynamic range, in addition to marker and panel optimization strategies. The streamlined workflow using off-the-shelf antibodies, data quality enabling downstream analysis, and ease of reaching hyperplex levels make seqIF suitable for immune-oncology research and other disciplines requiring spatial analysis, paving the way for its adoption in clinical settings. |
| first_indexed | 2024-03-09T15:11:15Z |
| format | Article |
| id | doaj.art-e3a417ff679f42828b038f7ea1871887 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-09T15:11:15Z |
| publishDate | 2023-10-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-e3a417ff679f42828b038f7ea18718872023-11-26T13:21:50ZengNature PortfolioScientific Reports2045-23222023-10-0113111410.1038/s41598-023-43435-wFully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomicsFrançois Rivest0Deniz Eroglu1Benjamin Pelz2Joanna Kowal3Alexandre Kehren4Vytautas Navikas5Maria Giuseppina Procopio6Pino Bordignon7Emilie Pérès8Marco Ammann9Emmanuel Dorel10Sylvain Scalmazzi11Lorenzo Bruno12Matthieu Ruegg13Gabriel Campargue14Gilles Casqueiro15Lionel Arn16Jérôme Fischer17Saska Brajkovic18Pierre Joris19Marco Cassano20Diego Dupouy21Lunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SALunaphore Technologies SAAbstract Tissues are complex environments where different cell types are in constant interaction with each other and with non-cellular components. Preserving the spatial context during proteomics analyses of tissue samples has become an important objective for different applications, one of the most important being the investigation of the tumor microenvironment. Here, we describe a multiplexed protein biomarker detection method on the COMET instrument, coined sequential ImmunoFluorescence (seqIF). The fully automated method uses successive applications of antibody incubation and elution, and in-situ imaging enabled by an integrated microscope and a microfluidic chip that provides optimized optical access to the sample. We show seqIF data on different sample types such as tumor and healthy tissue, including 40-plex on a single tissue section that is obtained in less than 24 h, using off-the-shelf antibodies. We also present extensive characterization of the developed method, including elution efficiency, epitope stability, repeatability and reproducibility, signal uniformity, and dynamic range, in addition to marker and panel optimization strategies. The streamlined workflow using off-the-shelf antibodies, data quality enabling downstream analysis, and ease of reaching hyperplex levels make seqIF suitable for immune-oncology research and other disciplines requiring spatial analysis, paving the way for its adoption in clinical settings.https://doi.org/10.1038/s41598-023-43435-w |
| spellingShingle | François Rivest Deniz Eroglu Benjamin Pelz Joanna Kowal Alexandre Kehren Vytautas Navikas Maria Giuseppina Procopio Pino Bordignon Emilie Pérès Marco Ammann Emmanuel Dorel Sylvain Scalmazzi Lorenzo Bruno Matthieu Ruegg Gabriel Campargue Gilles Casqueiro Lionel Arn Jérôme Fischer Saska Brajkovic Pierre Joris Marco Cassano Diego Dupouy Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics Scientific Reports |
| title | Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics |
| title_full | Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics |
| title_fullStr | Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics |
| title_full_unstemmed | Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics |
| title_short | Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics |
| title_sort | fully automated sequential immunofluorescence seqif for hyperplex spatial proteomics |
| url | https://doi.org/10.1038/s41598-023-43435-w |
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