Antibodies for tissue-based CyCIF

Tissue cyclic immunofluorescence (t-CyCIF) uses antibodies to spatially profile protein targets in human resection specimens. Commercially available antibodies that perform well in immunohistochemistry (IHC) and immunofluorescence (IF) assays are not guaranteed compatible with t-CyCIF, since tissue preparation, antigen retrieval, and processing protocols vary. It is therefore important that each antibody is specifically qualified for t-CyCIF.

At a minimum, antibodies should be tested in multiple tissue specimens, including positive and negative controls; tissue-microarrays (TMAs) are ideal in this regard. Fluorescently-conjugated antibodies may also be tested at a variety of concentrations to optimize signal-to-noise ratio. We report a detailed protocol for step-by-step procedures for qualifying antibodies in t-CyCIF imaging (Du et al. 2019).

We have tested a number of antibodies in formalin-fixed paraffin-embedded (FFPE) tissue specimens against different proteins for their compatibility with t-CyCIF. These include lineage makers, cytoskeletal proteins, cell cycle regulators, the phosphorylated forms of signaling proteins and kinases, transcription factors, and markers of cell state including quiescence, senescence, apoptosis, and stress. We report a select number of antibodies recommended for t-CyCIF below. While performance should not be equated to validation status, it provides some insight as to which antibodies have performed well in the past and are expected to perform well in new projects.

Last updated: February 2021

Last updated: February 2021

Archived Lists

  1. 2018 full table: The qualified list of 300 antibodies used in the Lin, et al. eLife 2018 paper

  2. 2018 recommended: Representative images of antibody staining patterns for a subset of 41 antibodies from the Lin, et al. eLife 2018 paper

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Funded by NIH grants P50-GM107618, U54-HL127365, U2C-CA233262, U2C-CA233280, U54-CA225088, the Ludwig Center at Harvard, and the Ludwig Cancer Research Foundation
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