免疫荧光
抗体
枚举
抗原
病理
免疫系统
计算生物学
生物
免疫学
医学
数学
组合数学
作者
Ziming Du,Jia‐Ren Lin,Rumana Rashid,Zoltan Maliga,Shu Wang,Jon C. Aster,Benjamin Izar,Peter K. Sorger,Sandro Santagata
出处
期刊:Nature Protocols
[Springer Nature]
日期:2019-09-18
卷期号:14 (10): 2900-2930
被引量:107
标识
DOI:10.1038/s41596-019-0206-y
摘要
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.
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