Authors:  Grady Carlson, PhD & Bethany Remeniuk, PhD


Our Phenoptics platform is now the established gold standard for advanced multiplex immunohistochemistry (IHC). The cornerstone of this technology is our Opalâ„¢ reagents, which utilize tyramide signal amplification (TSA) to increase antigen marker intensity by 10-100 fold compared to conventional indirect immunofluorescence methods. This enables practical and reliable multiplexed staining and analysis of up to eight markers plus DAPI counterstain on a single FFPE tissue section while reducing signal crosstalk and autofluorescence when used in conjunction with multispectral imaging. To assist you in your multiplex panel optimizations, we have created this table of published protocols, including primary antibodies, antigen retrieval buffer, and associated Opal fluorophore pairings (references listed below).

Primary antibodies used in Opal staining on FFPE tissue. Cell Signaling Technologies (CST); Coumarin (CMN, replaced by Polaris 480); Developmental Studies Hybridoma Bank (DSHB); Leica Biosystems (LB); Spring Biosciences (SB); Thermo Fisher Scientific (TFS); Ventana Medical Systems (VMS). *Compatible Opal fluorophores and dilutions.

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N. A. Giraldo et al. J Immunother Cancer 6, 99 (2018).
P. T. Nghiem et al. N Engl J Med 374, 2542-2552 (2016).
J. Lazarus et al. JCI Insight 3, (2018).
Z. Feng et al. JCI Insight 2, (2017).
J. L. Carstens et al.. Nature Communications 8, 15095 (2017).
M. A. J. Gorris et al. J Immunol 200, 347-354 (2018).