Qualifying Antibodies for Image-based Immune Profiling and Multiplexed Tissue Imaging

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2019 Sep 20

PMID 31534232

Citations 62

Authors

Ziming Du

Jia-Ren Lin

Rumana Rashid

Zoltan Maliga

Shu Wang

Jon C Aster

Benjamin Izar

Peter K Sorger

Sandro Santagata

Affiliations

Soon will be listed here.

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.

Citing Articles

Quantitative investigation of photobleaching-based autofluorescence suppression in formalin-fixed paraffin-embedded human tissue.

Hwang W, McPartland T, Raymond T, Han M, Jeong S, Evans C Biomed Opt Express. 2025; 16(2):553-565.

PMID: 39958863 PMC: 11828445. DOI: 10.1364/BOE.547033.

Personalized cancer vaccine design using AI-powered technologies.

Kumar A, Dixit S, Srinivasan K, M D, Vincent P Front Immunol. 2024; 15:1357217.

PMID: 39582860 PMC: 11581883. DOI: 10.3389/fimmu.2024.1357217.

Single-cell and spatial omics unravel the spatiotemporal biology of tumour border invasion and haematogenous metastasis.

Cheng X, Cao Y, Liu X, Li Y, Li Q, Gao D Clin Transl Med. 2024; 14(10):e70036.

PMID: 39350478 PMC: 11442492. DOI: 10.1002/ctm2.70036.

Basal-to-inflammatory transition and tumor resistance via crosstalk with a pro-inflammatory stromal niche.

Li N, Zhang W, Haensel D, Jussila A, Pan C, Gaddam S Nat Commun. 2024; 15(1):8134.

PMID: 39289380 PMC: 11408617. DOI: 10.1038/s41467-024-52394-3.

Fluorescence Lifetime Multiplexing (FLEX) for simultaneous high dimensional spatial biology in 3D.

Hwang W, Raymond T, McPartland T, Jeong S, Evans C Commun Biol. 2024; 7(1):1012.

PMID: 39154126 PMC: 11330493. DOI: 10.1038/s42003-024-06702-8.

References

Cyriac G, Gandhi L . Emerging biomarkers for immune checkpoint inhibition in lung cancer. Semin Cancer Biol. 2018; 52(Pt 2):269-277. DOI: 10.1016/j.semcancer.2018.05.006. View

Falcon B, Stewart J, Ezell S, Hanson J, Wijsman J, Ye X . High-content multiplexed tissue imaging and quantification for cancer drug discovery. Drug Discov Today. 2012; 18(11-12):510-22. DOI: 10.1016/j.drudis.2012.08.008. View

Bodenmiller B . Multiplexed Epitope-Based Tissue Imaging for Discovery and Healthcare Applications. Cell Syst. 2016; 2(4):225-38. DOI: 10.1016/j.cels.2016.03.008. View

Carvajal-Hausdorf D, Schalper K, Neumeister V, Rimm D . Quantitative measurement of cancer tissue biomarkers in the lab and in the clinic. Lab Invest. 2014; 95(4):385-96. PMC: 4383674. DOI: 10.1038/labinvest.2014.157. View

Giesen C, Wang H, Schapiro D, Zivanovic N, Jacobs A, Hattendorf B . Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat Methods. 2014; 11(4):417-22. DOI: 10.1038/nmeth.2869. View

Angelo M, Bendall S, Finck R, Hale M, Hitzman C, Borowsky A . Multiplexed ion beam imaging of human breast tumors. Nat Med. 2014; 20(4):436-42. PMC: 4110905. DOI: 10.1038/nm.3488. View

Keren L, Bosse M, Marquez D, Angoshtari R, Jain S, Varma S . A Structured Tumor-Immune Microenvironment in Triple Negative Breast Cancer Revealed by Multiplexed Ion Beam Imaging. Cell. 2018; 174(6):1373-1387.e19. PMC: 6132072. DOI: 10.1016/j.cell.2018.08.039. View

Glass G, Papin J, Mandell J . SIMPLE: a sequential immunoperoxidase labeling and erasing method. J Histochem Cytochem. 2009; 57(10):899-905. PMC: 2746723. DOI: 10.1369/jhc.2009.953612. View

Toth Z, Mezey E . Simultaneous visualization of multiple antigens with tyramide signal amplification using antibodies from the same species. J Histochem Cytochem. 2007; 55(6):545-54. DOI: 10.1369/jhc.6A7134.2007. View

10.

Pirici D, Mogoanta L, Kumar-Singh S, Pirici I, Margaritescu C, Simionescu C . Antibody elution method for multiple immunohistochemistry on primary antibodies raised in the same species and of the same subtype. J Histochem Cytochem. 2009; 57(6):567-75. PMC: 2690408. DOI: 10.1369/jhc.2009.953240. View

11.

Remark R, Merghoub T, Grabe N, Litjens G, Damotte D, Wolchok J . In-depth tissue profiling using multiplexed immunohistochemical consecutive staining on single slide. Sci Immunol. 2017; 1(1):aaf6925. PMC: 10152404. DOI: 10.1126/sciimmunol.aaf6925. View

12.

Wang Y, Woehrstein J, Donoghue N, Dai M, Avendano M, Schackmann R . Rapid Sequential in Situ Multiplexing with DNA Exchange Imaging in Neuronal Cells and Tissues. Nano Lett. 2017; 17(10):6131-6139. PMC: 5658129. DOI: 10.1021/acs.nanolett.7b02716. View

13.

Gerdes M, Sevinsky C, Sood A, Adak S, Bello M, Bordwell A . Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue. Proc Natl Acad Sci U S A. 2013; 110(29):11982-7. PMC: 3718135. DOI: 10.1073/pnas.1300136110. View

14.

Schubert W, Bonnekoh B, Pommer A, Philipsen L, Bockelmann R, Malykh Y . Analyzing proteome topology and function by automated multidimensional fluorescence microscopy. Nat Biotechnol. 2006; 24(10):1270-8. DOI: 10.1038/nbt1250. View

15.

Friedenberger M, Bode M, Krusche A, Schubert W . Fluorescence detection of protein clusters in individual cells and tissue sections by using toponome imaging system: sample preparation and measuring procedures. Nat Protoc. 2007; 2(9):2285-94. DOI: 10.1038/nprot.2007.320. View

16.

Hillert R, Gieseler A, Krusche A, Humme D, Rowert-Huber H, Sterry W . Large molecular systems landscape uncovers T cell trapping in human skin cancer. Sci Rep. 2016; 6:19012. PMC: 4725819. DOI: 10.1038/srep19012. View

17.

Lin J, Izar B, Wang S, Yapp C, Mei S, Shah P . Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes. Elife. 2018; 7. PMC: 6075866. DOI: 10.7554/eLife.31657. View

18.

Wahlby C, Erlandsson F, Bengtsson E, Zetterberg A . Sequential immunofluorescence staining and image analysis for detection of large numbers of antigens in individual cell nuclei. Cytometry. 2002; 47(1):32-41. View

19.

Stahl P, Salmen F, Vickovic S, Lundmark A, Fernandez Navarro J, Magnusson J . Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science. 2016; 353(6294):78-82. DOI: 10.1126/science.aaf2403. View

20.

Lee J, Daugharthy E, Scheiman J, Kalhor R, Yang J, Ferrante T . Highly multiplexed subcellular RNA sequencing in situ. Science. 2014; 343(6177):1360-3. PMC: 4140943. DOI: 10.1126/science.1250212. View