Opal staining system

Paraffin-embedded primary melanoma tissue samples were provided by UCT Frankfurt. Tumor sections (4 µm) were stained and analyzed using the Opal staining system according to the manufacturer’s instructions (PerkinElmer, Rodgau, Germany). The following antibodies were used for staining: IKKε (Active Motif, Carlsbad, CA, USA), TBK1 (Cell Signaling Technologies (Boston, MA, USA)), CD45 (Abcam, Cambridge, UK), PD1 (Abcam, Cambridge, UK), CD3 (Ventana, Roche Diagnostics, Mannheim, Germany). Corresponding secondary HRP-coupled antibodies were anti-mouse IgG-HRP and anti-rabbit IgG-HRP (both GE Healthcare, Freiburg, Germany). Nuclei were counterstained with DAPI and slides were mounted with Aqua-Poly/Mount (Polysciences, Hirschberg, Germany). The slides were imaged at 4× and 20× magnification using the Vectra3 imaging software, and the images were analyzed using the inForm2.0 Software (PerkinElmer, Rodgau, Germany).

Peritoneal membranes from mice at day 6 of the peritonitis model were fixed and paraffin embedded. Deparaffinized and rehydrated membrane sections (4 µm) were stained using the Opal staining system according to the manufacturer’s instructions (Perkin Elmer, Waltham, USA) with primary antibodies against CX3CL1 (MAB571, R&D Systems) and pan-cytokeratin (panCK) (ab7753; Abcam, Cambridge, UK). Pictures were acquired with the Vectra Polaris Automated Quantitative Pathology Imaging System featuring MOTiF (Akoya Biosciences, Marlborough, USA). The relative abundance of CX3CL1 expressing cells in the epithelium was scored upon tissue segmentation based on panCK staining with the InForm software (Akoya Biosciences).

Formaldehyde fixed, paraffin embedded sections were sequentially stained with antibodies against mouse CD163 (Abcam, #ab182422; 1:250 dilution), CD31 (Cell Signaling, #77699; 1:500 dilution), and LYVE-1 (R&D Systems, #AF2125; 1:200 dilution) using the Opal staining system according to the manufacturer’s instructions (Perkin Elmer). Nuclei were counterstained with DAPI and slides were mounted with Fluoromount-G (SouthernBiotech, Birmingham, USA). The Vectra^® 3 automated quantitative pathology imaging system (PerkinElmer, Rodgau, Germany) was used for image acquisition at 20× and images were analyzed using the inForm2.0 Software (PerkinElmer) and ImageJ as follows. Composite images of whole tissue sections were RGB stacked. The threshold of the DAPI signal was set for positive identification of all cells in the frame. Set threshold values were used for quantification of pixels in each channel. Data are presented as percentage area occupied by stain of interest in all images of a tissue by DAPI stain.