Simple, flexible and robust

CyCIF is a robust and inexpensive method for highly multiplexed immunofluorescence imaging using standard instruments and reagents. The concept of repeatedly staining and imaging slides has been around for many years and most commonly involves antibody stripping using denaturants. Gerdes et al (2013) described an approach in which fluorophores are chemically inactivated after each of several rounds of immunofluorescence. The t-CyCIF method by Lin et al (2018) builds on this and related approaches. The rich data collected using CyCIF are also amenable to state-of-the-art, high-dimensionality analysis tools developed for CyTOF, including tSNE , viSNE and Wanderlust. These allow investigation of complex associations and interdependencies between observed features and phenotypes.

Tumor ecosystem in its preserved architecture

t-CyCIF (Tissue-based cyclic immunofluorescence) can be used to quantify signal transduction cascades, measure the levels of tumor antigens and determine immunophenotypes using immune cell lineage markers. t-CyCIF is a powerful tool to study drug resistance of immunotherapy in different patients.


t-CyCIF (Tissue-based cyclic immunofluorescence) uses formalin-fixed, paraffin-embedded (FFPE) tumor and tissue specimens mounted on glass slides. These are the most widely used specimens for histopathological diagnosis of cancer and other diseases. t-CyCIF generates multiplexed images of FFPE samples using an iterative process (a cycle) in which conventional low-plex fluorescence images are repeatedly collected from the same sample and then assembled into a high dimensional representation. Several variants are possible using direct and indirect immunofluorescence.

Key features of t-CyCIF

  • Works with a wide range of FFPE human and mouse tumors and tissues.
  • Up to 60-plex imaging of some tissues — compatible with H&E in final round.
  • Uses conventional wide-field, confocal and super-resolution microscopes.
  • Antibodies can be selected based on the requirements of the project: no proprietary or unusual reagents required.