Bond polymer refine

Formalin-fixed, paraffin-embedded (FFPE) tissues from normal and tumour samples were included in several TMA blocks. IHC analyses and in situ hybridization for the Epstein–Barr virus (EBV) were performed on TMAs using heat-induced epitope retrieval and standard procedures. Antibodies sources and dilution are described in Table B in S1 File. Protein expression was quantified using an automated scan, Chroma Vision Systems- ACIS III (DAKO, Glostrup, Denmark) as previously described [13 (link)]. For survival analyses, all IHC markers were categorized as high or low, using the median expression as the cut-off.
The Bond Polymer Refine detection system (Leica Biosystems, Solms, Germany) was used for single and double immunoenzymatic labelling of FFPE tissues, using several normal lymphoid tissues and 10 cases of cHL. Double labelling, using a double immunoperoxidase technique or the immunoperoxidase technique combined with immunofluorescence, was also performed to assess the relationship between CSF1R+, CD68+, CD163+ and CD30+ cells. Complete details of the protocol and antibodies are available in S1 File.

Sections from tissue blocks of all cases were immunohistochemically stained with STING (anti-TMEM173; clone SP338, dilution 1:150; Abcam, UK). Heat-induced antigen retrieval for STING was performed using a microwave oven and 0.01mol/L of citrate buffer, pH 8.0, for 30 min. Moreover, tumors defined as “excluded” and “inflamed” by hematoxylin and eosin were investigated for the specific composition of different lymphocytes subpopulations, according to the different percentages of expression of the following antibodies: CD3 (clone PS1, dilution 1:200, LEICA), CD20 (clone L26, prediluted, NOVOCASTRA), CD4 (clone 4B12, dilution 1.150, LEICA), CD8 (clone 29S, dilution 1:20, LEICA), and FOXP3 (clone 221D/D3, dilution 1:200, SEROTEC). All samples were processed using a sensitive “Bond Polymer Refine” detection system in an automated bond immunohistochemistry instrument (Leica Biosystems, Germany). Sections incubated without the primary antibody served as a negative control. Cytoplasmic and membranous labeling for the STING was recorded by combining the percentage of positive cells (0–100%) multiplied by staining intensity (0, 1+, 2+, and 3+) to obtain an overall H-score (0-300).