CODEX multiplexed tissue imaging with DNA-conjugated antibodies
Advances in multiplexed imaging technologies have drastically improved our ability to characterize healthy and diseased tissues at the single-cell level. Co-detection by indexing (CODEX) relies on DNA-conjugated antibodies and the cyclic addition and removal of complementary fluorescently labeled DN...
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| Veröffentlicht in: | Nature protocols 2021-08, Vol.16 (8), p.3802-3835 |
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| creator | Black, Sarah Phillips, Darci Hickey, John W. Kennedy-Darling, Julia Venkataraaman, Vishal G. Samusik, Nikolay Goltsev, Yury Schürch, Christian M. Nolan, Garry P. |
| description | Advances in multiplexed imaging technologies have drastically improved our ability to characterize healthy and diseased tissues at the single-cell level. Co-detection by indexing (CODEX) relies on DNA-conjugated antibodies and the cyclic addition and removal of complementary fluorescently labeled DNA probes and has been used so far to simultaneously visualize up to 60 markers in situ. CODEX enables a deep view into the single-cell spatial relationships in tissues and is intended to spur discovery in developmental biology, disease and therapeutic design. Herein, we provide optimized protocols for conjugating purified antibodies to DNA oligonucleotides, validating the conjugation by CODEX staining and executing the CODEX multicycle imaging procedure for both formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissues. In addition, we describe basic image processing and data analysis procedures. We apply this approach to an FFPE human tonsil multicycle experiment. The hands-on experimental time for antibody conjugation is ~4.5 h, validation of DNA-conjugated antibodies with CODEX staining takes ~6.5 h and preparation for a CODEX multicycle experiment takes ~8 h. The multicycle imaging and data analysis time depends on the tissue size, number of markers in the panel and computational complexity.
This protocol describes co-detection by indexing, a highly multiplexed imaging technology that uses DNA-conjugated antibodies to image up to 60 markers in formalin-fixed, paraffin-embedded and fresh-frozen tissues. |
| doi_str_mv | 10.1038/s41596-021-00556-8 |
| format | Article |
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This protocol describes co-detection by indexing, a highly multiplexed imaging technology that uses DNA-conjugated antibodies to image up to 60 markers in formalin-fixed, paraffin-embedded and fresh-frozen tissues.</description><identifier>ISSN: 1754-2189</identifier><identifier>ISSN: 1750-2799</identifier><identifier>EISSN: 1750-2799</identifier><identifier>DOI: 10.1038/s41596-021-00556-8</identifier><identifier>PMID: 34215862</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>631/1647 ; 631/250 ; 631/61 ; 631/67 ; 692/420 ; Analysis ; Analytical Chemistry ; Animals ; Antibodies ; Antibodies - chemistry ; Biological Techniques ; Biomarkers ; Biomedical and Life Sciences ; Computational Biology/Bioinformatics ; Computer applications ; Conjugation ; Data analysis ; Deoxyribonucleic acid ; Design optimization ; Developmental biology ; Diagnostic Imaging ; Diagnostic specimens ; DNA ; DNA - chemistry ; DNA probes ; Equipment and supplies ; Fluorescence microscopy ; Fluorescent indicators ; Formaldehyde ; Haplorhini ; Humans ; Image processing ; Image Processing, Computer-Assisted ; Indexing ; Life Sciences ; Markers ; Methods ; Mice ; Microarrays ; Multiplexing ; Oligonucleotides ; Organic Chemistry ; Paraffin ; Paraffin Embedding ; Paraffins ; Protocol ; Reproducibility of Results ; Single-Cell Analysis - methods ; Staining ; Tissue Fixation - methods ; Tissues ; Tonsil ; Viral antibodies</subject><ispartof>Nature protocols, 2021-08, Vol.16 (8), p.3802-3835</ispartof><rights>The Author(s), under exclusive licence to Springer Nature Limited 2021</rights><rights>2021. The Author(s), under exclusive licence to Springer Nature Limited.</rights><rights>COPYRIGHT 2021 Nature Publishing Group</rights><rights>The Author(s), under exclusive licence to Springer Nature Limited 2021.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c575t-5592a50c9ca9a1ae2fae7cb998c868172d9f4d1c47dd9a068d72ac8b3025cf423</citedby><cites>FETCH-LOGICAL-c575t-5592a50c9ca9a1ae2fae7cb998c868172d9f4d1c47dd9a068d72ac8b3025cf423</cites><orcidid>0000-0002-8862-9043 ; 0000-0002-1792-1768 ; 0000-0002-0804-3342</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,780,784,885,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/34215862$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Black, Sarah</creatorcontrib><creatorcontrib>Phillips, Darci</creatorcontrib><creatorcontrib>Hickey, John W.</creatorcontrib><creatorcontrib>Kennedy-Darling, Julia</creatorcontrib><creatorcontrib>Venkataraaman, Vishal G.</creatorcontrib><creatorcontrib>Samusik, Nikolay</creatorcontrib><creatorcontrib>Goltsev, Yury</creatorcontrib><creatorcontrib>Schürch, Christian M.</creatorcontrib><creatorcontrib>Nolan, Garry P.</creatorcontrib><title>CODEX multiplexed tissue imaging with DNA-conjugated antibodies</title><title>Nature protocols</title><addtitle>Nat Protoc</addtitle><addtitle>Nat Protoc</addtitle><description>Advances in multiplexed imaging technologies have drastically improved our ability to characterize healthy and diseased tissues at the single-cell level. Co-detection by indexing (CODEX) relies on DNA-conjugated antibodies and the cyclic addition and removal of complementary fluorescently labeled DNA probes and has been used so far to simultaneously visualize up to 60 markers in situ. CODEX enables a deep view into the single-cell spatial relationships in tissues and is intended to spur discovery in developmental biology, disease and therapeutic design. Herein, we provide optimized protocols for conjugating purified antibodies to DNA oligonucleotides, validating the conjugation by CODEX staining and executing the CODEX multicycle imaging procedure for both formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissues. In addition, we describe basic image processing and data analysis procedures. We apply this approach to an FFPE human tonsil multicycle experiment. The hands-on experimental time for antibody conjugation is ~4.5 h, validation of DNA-conjugated antibodies with CODEX staining takes ~6.5 h and preparation for a CODEX multicycle experiment takes ~8 h. The multicycle imaging and data analysis time depends on the tissue size, number of markers in the panel and computational complexity.
This protocol describes co-detection by indexing, a highly multiplexed imaging technology that uses DNA-conjugated antibodies to image up to 60 markers in formalin-fixed, paraffin-embedded and fresh-frozen tissues.</description><subject>631/1647</subject><subject>631/250</subject><subject>631/61</subject><subject>631/67</subject><subject>692/420</subject><subject>Analysis</subject><subject>Analytical Chemistry</subject><subject>Animals</subject><subject>Antibodies</subject><subject>Antibodies - chemistry</subject><subject>Biological Techniques</subject><subject>Biomarkers</subject><subject>Biomedical and Life Sciences</subject><subject>Computational Biology/Bioinformatics</subject><subject>Computer applications</subject><subject>Conjugation</subject><subject>Data analysis</subject><subject>Deoxyribonucleic acid</subject><subject>Design optimization</subject><subject>Developmental biology</subject><subject>Diagnostic Imaging</subject><subject>Diagnostic specimens</subject><subject>DNA</subject><subject>DNA - 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chemistry</topic><topic>Biological Techniques</topic><topic>Biomarkers</topic><topic>Biomedical and Life Sciences</topic><topic>Computational Biology/Bioinformatics</topic><topic>Computer applications</topic><topic>Conjugation</topic><topic>Data analysis</topic><topic>Deoxyribonucleic acid</topic><topic>Design optimization</topic><topic>Developmental biology</topic><topic>Diagnostic Imaging</topic><topic>Diagnostic specimens</topic><topic>DNA</topic><topic>DNA - chemistry</topic><topic>DNA probes</topic><topic>Equipment and supplies</topic><topic>Fluorescence microscopy</topic><topic>Fluorescent indicators</topic><topic>Formaldehyde</topic><topic>Haplorhini</topic><topic>Humans</topic><topic>Image processing</topic><topic>Image Processing, Computer-Assisted</topic><topic>Indexing</topic><topic>Life Sciences</topic><topic>Markers</topic><topic>Methods</topic><topic>Mice</topic><topic>Microarrays</topic><topic>Multiplexing</topic><topic>Oligonucleotides</topic><topic>Organic Chemistry</topic><topic>Paraffin</topic><topic>Paraffin Embedding</topic><topic>Paraffins</topic><topic>Protocol</topic><topic>Reproducibility of Results</topic><topic>Single-Cell Analysis - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Nature protocols</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Black, Sarah</au><au>Phillips, Darci</au><au>Hickey, John W.</au><au>Kennedy-Darling, Julia</au><au>Venkataraaman, Vishal G.</au><au>Samusik, Nikolay</au><au>Goltsev, Yury</au><au>Schürch, Christian M.</au><au>Nolan, Garry P.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>CODEX multiplexed tissue imaging with DNA-conjugated antibodies</atitle><jtitle>Nature protocols</jtitle><stitle>Nat Protoc</stitle><addtitle>Nat Protoc</addtitle><date>2021-08-01</date><risdate>2021</risdate><volume>16</volume><issue>8</issue><spage>3802</spage><epage>3835</epage><pages>3802-3835</pages><issn>1754-2189</issn><issn>1750-2799</issn><eissn>1750-2799</eissn><abstract>Advances in multiplexed imaging technologies have drastically improved our ability to characterize healthy and diseased tissues at the single-cell level. Co-detection by indexing (CODEX) relies on DNA-conjugated antibodies and the cyclic addition and removal of complementary fluorescently labeled DNA probes and has been used so far to simultaneously visualize up to 60 markers in situ. CODEX enables a deep view into the single-cell spatial relationships in tissues and is intended to spur discovery in developmental biology, disease and therapeutic design. Herein, we provide optimized protocols for conjugating purified antibodies to DNA oligonucleotides, validating the conjugation by CODEX staining and executing the CODEX multicycle imaging procedure for both formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissues. In addition, we describe basic image processing and data analysis procedures. We apply this approach to an FFPE human tonsil multicycle experiment. The hands-on experimental time for antibody conjugation is ~4.5 h, validation of DNA-conjugated antibodies with CODEX staining takes ~6.5 h and preparation for a CODEX multicycle experiment takes ~8 h. The multicycle imaging and data analysis time depends on the tissue size, number of markers in the panel and computational complexity.
This protocol describes co-detection by indexing, a highly multiplexed imaging technology that uses DNA-conjugated antibodies to image up to 60 markers in formalin-fixed, paraffin-embedded and fresh-frozen tissues.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>34215862</pmid><doi>10.1038/s41596-021-00556-8</doi><tpages>34</tpages><orcidid>https://orcid.org/0000-0002-8862-9043</orcidid><orcidid>https://orcid.org/0000-0002-1792-1768</orcidid><orcidid>https://orcid.org/0000-0002-0804-3342</orcidid><oa>free_for_read</oa></addata></record> |
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| language | eng |
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| subjects | 631/1647 631/250 631/61 631/67 692/420 Analysis Analytical Chemistry Animals Antibodies Antibodies - chemistry Biological Techniques Biomarkers Biomedical and Life Sciences Computational Biology/Bioinformatics Computer applications Conjugation Data analysis Deoxyribonucleic acid Design optimization Developmental biology Diagnostic Imaging Diagnostic specimens DNA DNA - chemistry DNA probes Equipment and supplies Fluorescence microscopy Fluorescent indicators Formaldehyde Haplorhini Humans Image processing Image Processing, Computer-Assisted Indexing Life Sciences Markers Methods Mice Microarrays Multiplexing Oligonucleotides Organic Chemistry Paraffin Paraffin Embedding Paraffins Protocol Reproducibility of Results Single-Cell Analysis - methods Staining Tissue Fixation - methods Tissues Tonsil Viral antibodies |
| title | CODEX multiplexed tissue imaging with DNA-conjugated antibodies |
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