Multiplex Immunofluorescence Phenotyping of CICs

The “EML” method was used to subtype CICs as previously reported (9 (link)). In brief, samples were first stained with antibody against CD45 (mouse mAb from Boster, BM0091) at a dilution of 1:400 by Opal Multiplex tissue staining kit (Perkin Elmer, NEL791001KT) according to the standard protocol provided, and CD45 molecules were eventually labeled with Cyanine 5 fluorophore. Slides were then incubated with mixed antibodies against E-cadherin (mouse mAb from BD Biosciences, 610181) and CD68 (rabbit pAb from Proteintech, 25747–1-AP), followed by secondary antibodies of Alexa Fluor 568 anti-rabbit antibody (Invitrogen, A11036) and Alexa Fluor 488 anti-mouse antibody (Invitrogen, A11029). All slides were counterstained with DAPI to show nuclei and mounted with Antifade reagent (Invitrogen, Carlsbad, CA) and cover slips.
Multispectral images were taken with TMA modules of Vectra® Automated Imaging System (Perkin Elmer) by a 20× objective lens. Nuance system (Perkin Elmer) was used to build libraries of each spectrum (DAPI, FITC, TRITC, and Cy5) and unmix multispectral images with high contrast and accuracy. InForm automated image analysis software (Perkin Elmer) was used for batch analysis of multispectral images based on specified algorithms.