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Multiplex Immunofluorescence of tumor biomarkers in breast cancer tumor microenvironment. Multiplex immunofluorescence staining of formaldehyde fixed paraffin embedded tumor slide specimens of four breast cancer subtypes were performed using Opal IHC Multiplex Assay kit (NEL821001KT, Akoya Biosciences) according to the manufacturer’s protocol. Sections were sequentially stained as follows: MUC16/CA125 (clone EPR1020(2), dilution 1:1000, Abcam), MUC1/CA153 (clone EPR1023, dilution 1:500, Abcam), CEA (clone EPCEAR7, dilution 1:2000, Abcam), and SEMA4C (28402-1-AP, dilution 1:500, Proteintech). Briefly, slides were microwave heat-treated in Epitope retrieval solution 1 or 2 buffer, blocked in Antibody Diluent, and incubated with the primary antibody, HRP-conjugated secondary polymer, OPAL fluorescent reagents, and DAPI. Whole slide scans were acquired using the Vectra3 automated quantitative pathology imaging system (Akoya Biosciences).
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