Opal multiplex fihc kit

mIHC/IF was performed using an Opal Multiplex fIHC kit (PerkinElmer, Inc, Waltham, Massachusetts, USA), as previously described by our group and in other studies.25 31–52 (link) Tissue sections (4 µm thick) were labeled with primary antibodies against CD38, CD8 and CD68, followed by appropriate secondary antibodies. All antibodies used are listed in online supplementary table 2. This was followed by the application of a fluorophore-conjugated tyramide signal amplification buffer (PerkinElmer, Inc) and the nuclear counterstain DAPI. A Vectra three pathology imaging system microscope (PerkinElmer, Inc) was used to obtain images, and these were analyzed using inForm software (V.2.4.2; PerkinElmer, Inc)34 47 53 54 (link) and HALO TM (Indica Labs, Albuquerqe, New Mexico, USA).55–59 (link)
The density of CD38⁺CD68⁺ macrophages and CD8⁺ T cells were determined as follows: cell count per predefined, high-powered field (334 μm × 250 μm) represents the density of CD38⁺CD68⁺ macrophages and CD8⁺ T cells in the TME. Samples were then categorized as ‘high’ or ‘low’ according to whether the CD38⁺CD68⁺ macrophage and CD8⁺ T cell count was above the cut-off points (best thresholds) that produced the lowest p value, which were determined using previously described methods.25 31 32 36 37 43–52 60 61 (link)

Multiplex immunofluorescence was performed using an Opal Multiplex fIHC kit (PerkinElmer, Inc., Waltham, MA, USA), on FFPE tissue sections processed according to the standard immunohistochemistry protocol. Slides were incubated with primary antibodies against cytokeratin (CK), CD14, CD206, GM-CSF, and TNFα (as presented in Table S1), followed by appropriate secondary antibodies, before application of the fluorophore-conjugated tyramides signal amplification buffer (PerkinElmer, Inc., Waltham, MA, USA). DAPI was used as a nuclear counterstain, and images were acquired using a Vectra 3 pathology imaging system microscope (PerkinElmer, Inc.) and analysed using inForm version 2.3 software (PerkinElmer, Inc.). Mean intensities of each stain were determined in the nucleus, cytoplasm and membrane of individual cells delineated in each image. For DAPI, nucleus quantification was used but for the other stainings, cytoplasmic quantification was used rather than membrane quantification to limit contaminating signals coming from neighbouring cells. Data were next analysed by histo-cytometry using the FlowJo software (Tree Star). Merged images of CD14 and GM-CSF co-stainings were obtained using the Image J 1.51m9 software.