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

Nat Protoc. 2019 Oct;14(10):2900-2930. doi: 10.1038/s41596-019-0206-y. Epub 2019 Sep 18.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies*
  • Biomarkers, Tumor / immunology
  • Fluorescent Antibody Technique / instrumentation
  • Fluorescent Antibody Technique / methods*
  • Formaldehyde
  • Humans
  • Image Processing, Computer-Assisted / methods*
  • Immunohistochemistry / methods
  • Mice
  • Neoplasms / immunology
  • Neoplasms / pathology*
  • Paraffin Embedding


  • Antibodies
  • Biomarkers, Tumor
  • Formaldehyde