Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging

Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on...

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Bibliographic Details
Published inNature protocols Vol. 14; no. 10; pp. 2900 - 2930
Main Authors Du, Ziming, Lin, Jia-Ren, Rashid, Rumana, Maliga, Zoltan, Wang, Shu, Aster, Jon C., Izar, Benjamin, Sorger, Peter K., Santagata, Sandro
Format Journal Article
LanguageEnglish
Published London Nature Publishing Group UK 01.10.2019
Nature Publishing Group
Subjects
631/1647/245/2225
631/250/580
631/553/2706
631/67/327
692/53
Analytical Chemistry
Animals
Antibodies
Antigens
Biological Techniques
Biomarkers, Tumor - immunology
Biomedical and Life Sciences
Computational Biology/Bioinformatics
Data analysis
Enumeration
Fluorescence
Fluorescent antibody technique
Fluorescent Antibody Technique - instrumentation
Fluorescent Antibody Technique - methods
Formaldehyde
Health aspects
Humans
Image Processing, Computer-Assisted - methods
Imaging
Immune checkpoint
Immune system
Immunofluorescence
Immunoglobulins
Immunohistochemistry - methods
Immunological research
Life Sciences
Lymphocytes
Lymphocytes B
Lymphocytes T
Macrophages
Mapping
Measurement
Mice
Microarrays
Multiplexing
Neoplasms - immunology
Neoplasms - pathology
Organic Chemistry
Paraffin
Paraffin Embedding
Paraffins
Protocol
Regulators
Selectivity
Test procedures
Viral antibodies
United States
Online AccessGet full text
ISSN1754-2189
1750-2799
1750-2799
DOI10.1038/s41596-019-0206-y

Cover

Abstract Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks. This protocol provides guidelines for designing and validating antibody panels for fluorescence-based imaging of FFPE tissue sections using cyclic immunofluorescence (t-CyCIF) or other multiplexed imaging methods.
AbstractList Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks.
Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks. This protocol provides guidelines for designing and validating antibody panels for fluorescence-based imaging of FFPE tissue sections using cyclic immunofluorescence (t-CyCIF) or other multiplexed imaging methods.
Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks. This protocol provides guidelines for designing and validating antibody panels for fluorescence-based imaging of FFPE tissue sections using cyclic immunofluorescence (t-CyCIF) or other multiplexed imaging methods.
Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks. Further information on research design is available in the Nature Research Reporting Summary.
This protocol provides guidelines for designing and validating antibody panels for fluorescence-based imaging of FFPE tissue sections using cyclic immunofluorescence (t-CyCIF) or other multiplexed imaging methods.
Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks.Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks.
Audience Academic
Author Rashid, Rumana
Lin, Jia-Ren
Aster, Jon C.
Sorger, Peter K.
Wang, Shu
Izar, Benjamin
Maliga, Zoltan
Du, Ziming
Santagata, Sandro
AuthorAffiliation 2 Ludwig Center for Cancer Research at Harvard, Harvard Medical School, Boston, MA, USA
9 Department of Oncologic Pathology, Dana-Farber Cancer Institute, Boston, MA, USA
1 Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
4 Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA
11 These authors contributed equally: Ziming Du, Jia-Ren Lin, Rumana Rashid
10 Department of Pathology, Boston Children’s Hospital, Boston, MA, USA
6 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
7 Broad Institute of MIT and Harvard, Boston, MA, USA
5 Harvard Graduate Program in Biophysics, Harvard University, Boston, MA, USA
8 Department of Systems Biology, Harvard Medical School, Boston, MA, USA
3 Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA, USA
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BackLink https://www.ncbi.nlm.nih.gov/pubmed/31534232$$D View this record in MEDLINE/PubMed
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ContentType Journal Article
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Snippet Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A...
This protocol provides guidelines for designing and validating antibody panels for fluorescence-based imaging of FFPE tissue sections using cyclic...
Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A...
SourceID pubmedcentral
proquest
gale
pubmed
crossref
springer
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Index Database
Enrichment Source
Publisher
StartPage 2900
SubjectTerms 631/1647/245/2225
631/250/580
631/553/2706
631/67/327
692/53
Analytical Chemistry
Animals
Antibodies
Antigens
Biological Techniques
Biomarkers, Tumor - immunology
Biomedical and Life Sciences
Computational Biology/Bioinformatics
Data analysis
Enumeration
Fluorescence
Fluorescent antibody technique
Fluorescent Antibody Technique - instrumentation
Fluorescent Antibody Technique - methods
Formaldehyde
Health aspects
Humans
Image Processing, Computer-Assisted - methods
Imaging
Immune checkpoint
Immune system
Immunofluorescence
Immunoglobulins
Immunohistochemistry - methods
Immunological research
Life Sciences
Lymphocytes
Lymphocytes B
Lymphocytes T
Macrophages
Mapping
Measurement
Mice
Microarrays
Multiplexing
Neoplasms - immunology
Neoplasms - pathology
Organic Chemistry
Paraffin
Paraffin Embedding
Paraffins
Protocol
Regulators
Selectivity
Test procedures
Viral antibodies
Title Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging
URI https://link.springer.com/article/10.1038/s41596-019-0206-y
https://www.ncbi.nlm.nih.gov/pubmed/31534232
https://www.proquest.com/docview/2298214442
https://www.proquest.com/docview/2293981763
https://pubmed.ncbi.nlm.nih.gov/PMC6959005
Volume 14
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