A rapid method for multispectral fluorescence imaging of frozen tissue sections

Dinesh Jaishankar, Cormac Cosgrove, Ryan J. Deaton, I. Caroline Le Poole*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

Original languageEnglish (US)
Article numbere60806
JournalJournal of Visualized Experiments
Issue number157
StatePublished - Mar 2020


  • Cancer
  • Frozen tissues
  • Immune profiling
  • Immunology and Infection
  • Issue 157
  • Multiplexing
  • Multispectral imaging
  • Quantitative pathology

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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