Simultaneous amplification of multiple immunofluorescence signals via cyclic staining of target molecules using mutually cross-adsorbed antibodies
Abstract Amplification of immunofluorescence (IF) signals is becoming increasingly critical in cancer research and neuroscience. Recently, we put forward a new signal amplification technique, which we termed fluorescent signal amplification via cyclic staining of target molecules (FRACTAL). FRACTAL...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-05-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-12808-y |
| _version_ | 1811258840551784448 |
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| author | Haemin Yeon Yehlin Cho Junyoung Seo Yeonbo Sim Jae-Byum Chang |
| author_facet | Haemin Yeon Yehlin Cho Junyoung Seo Yeonbo Sim Jae-Byum Chang |
| author_sort | Haemin Yeon |
| collection | DOAJ |
| description | Abstract Amplification of immunofluorescence (IF) signals is becoming increasingly critical in cancer research and neuroscience. Recently, we put forward a new signal amplification technique, which we termed fluorescent signal amplification via cyclic staining of target molecules (FRACTAL). FRACTAL amplifies IF signals by repeatedly labeling target proteins with a pair of secondary antibodies that bind to each other. However, simultaneous amplification of multiple IF signals via FRACTAL has not yet been demonstrated because of cross-reactivity between the secondary antibodies. In this study, we show that mutual cross-adsorption between antibodies can eliminate all forms of cross-reactions between them, enabling simultaneous amplification of multiple IF signals. First, we show that a typical cross-adsorption process—in which an antibody binds to proteins with potential cross-reactivity with the antibody—cannot eliminate cross-reactions between antibodies in FRACTAL. Next, we show that all secondary antibodies used in FRACTAL need to be mutually cross-adsorbed to eliminate all forms of cross-reactivity, and then we demonstrate simultaneous amplification of multiple IF signals using these antibodies. Finally, we show that multiplexed FRACTAL can be applied to expansion microscopy to achieve higher fluorescence intensities after expansion. Multiplexed FRACTAL is a highly versatile tool for standard laboratories, as it amplifies multiple IF signals without the need for custom antibodies. |
| first_indexed | 2024-04-12T18:20:30Z |
| format | Article |
| id | doaj.art-4876c5a7e2a240ce8e4cbe23f73439e0 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-12T18:20:30Z |
| publishDate | 2022-05-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-4876c5a7e2a240ce8e4cbe23f73439e02022-12-22T03:21:26ZengNature PortfolioScientific Reports2045-23222022-05-0112111010.1038/s41598-022-12808-ySimultaneous amplification of multiple immunofluorescence signals via cyclic staining of target molecules using mutually cross-adsorbed antibodiesHaemin Yeon0Yehlin Cho1Junyoung Seo2Yeonbo Sim3Jae-Byum Chang4Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST)Department of Materials Science and Engineering, Massachusetts Institute of Technology (MIT)Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST)Department of Biomedical Engineering, Sungkyunkwan UniversityDepartment of Materials Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST)Abstract Amplification of immunofluorescence (IF) signals is becoming increasingly critical in cancer research and neuroscience. Recently, we put forward a new signal amplification technique, which we termed fluorescent signal amplification via cyclic staining of target molecules (FRACTAL). FRACTAL amplifies IF signals by repeatedly labeling target proteins with a pair of secondary antibodies that bind to each other. However, simultaneous amplification of multiple IF signals via FRACTAL has not yet been demonstrated because of cross-reactivity between the secondary antibodies. In this study, we show that mutual cross-adsorption between antibodies can eliminate all forms of cross-reactions between them, enabling simultaneous amplification of multiple IF signals. First, we show that a typical cross-adsorption process—in which an antibody binds to proteins with potential cross-reactivity with the antibody—cannot eliminate cross-reactions between antibodies in FRACTAL. Next, we show that all secondary antibodies used in FRACTAL need to be mutually cross-adsorbed to eliminate all forms of cross-reactivity, and then we demonstrate simultaneous amplification of multiple IF signals using these antibodies. Finally, we show that multiplexed FRACTAL can be applied to expansion microscopy to achieve higher fluorescence intensities after expansion. Multiplexed FRACTAL is a highly versatile tool for standard laboratories, as it amplifies multiple IF signals without the need for custom antibodies.https://doi.org/10.1038/s41598-022-12808-y |
| spellingShingle | Haemin Yeon Yehlin Cho Junyoung Seo Yeonbo Sim Jae-Byum Chang Simultaneous amplification of multiple immunofluorescence signals via cyclic staining of target molecules using mutually cross-adsorbed antibodies Scientific Reports |
| title | Simultaneous amplification of multiple immunofluorescence signals via cyclic staining of target molecules using mutually cross-adsorbed antibodies |
| title_full | Simultaneous amplification of multiple immunofluorescence signals via cyclic staining of target molecules using mutually cross-adsorbed antibodies |
| title_fullStr | Simultaneous amplification of multiple immunofluorescence signals via cyclic staining of target molecules using mutually cross-adsorbed antibodies |
| title_full_unstemmed | Simultaneous amplification of multiple immunofluorescence signals via cyclic staining of target molecules using mutually cross-adsorbed antibodies |
| title_short | Simultaneous amplification of multiple immunofluorescence signals via cyclic staining of target molecules using mutually cross-adsorbed antibodies |
| title_sort | simultaneous amplification of multiple immunofluorescence signals via cyclic staining of target molecules using mutually cross adsorbed antibodies |
| url | https://doi.org/10.1038/s41598-022-12808-y |
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