Opal multiplex tissue staining kit

The TMA slide was routinely de-paraffinized using the xylene-ethanol method following baking in 65°C for 2 h. Antigen retrieval was performed in citrate acid buffer by microwaving for 15 min after boiling, followed by 1 h of blocking in 5% bovine serum albumin in Tris-buffered saline. The slide was first stained with antibodies against CD45 (Boster, Wuhan, China; BM0091) using an Opal Multiplex tissue staining kit (Perkin Elmer, Waltham, MA, USA; NEL791001KT) according to the manufacturer’s instructions, and CD45 molecules were labeled with the Cyanine 5 fluorophore. Then, the slide was incubated with mixed antibodies against E-cadherin (BD Biosciences, San Jose, CA, USA; 610181) and CD44 (Abcam, Cambridge, MA, USA; 243894), followed by secondary antibodies comprising Alexa Fluor 488 anti-mouse antibodies (ThermoFisher, Waltham, MA, USA; A31561) and Alexa Fluor 564 anti-rabbit antibody (ThermoFisher, A11035). Samples were labeled with 4′,6-diamidino-2-phenylindole (DAPI) to show nuclei, followed by mounting with Antifade reagent (ThermoFisher, P36934), adding cover slips, and sealing with nail varnish. Images were acquired using the TMA modules of Vectra^® Automated Imaging System (Perkin Elmer) with a 20× objective lens.

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.