Vectra

Whole tissue section images were acquired at 200 × magnification using the imaging system Vectra (PerkinElmer, Waltham, MA, USA) as previously described [19] (link) and then images taken were analyzed using the image analysis software inForm (PerkinElmer) to enumerate the total number of CD68⁺ cells per tissue section. The macrophage infiltration (CD68⁺%) was expressed by dividing the cell number of CD68⁺ cells with the number of total cells of the tissue section.

Immunohistochemistry staining was conducted as indicated in the Ultra View Universal DAB Detection kit (Ventana Medical Systems, Inc., Oro Valley, AZ, USA). The slides were incubated at 60 °C for 4 min, Ezprep was treated for deparaffinization, and slides were rinsed with a Tris buffer (pH 7.6). After reacting the slides to the antigen retrieval buffer for 1 h at 100 °C, they were treated with rabbit anti-AhR (1:1000, Enzo Life Sciences, NY, USA) for 36 min at 37 °C. The rinsed slides were processed using the DISCOVERY Ultra Map antI/Rabbit HRP for 12 min at 37 °C. Ultraview DAB and DAB H₂O₂ were treated for 8 min at 37 °C, during which AhR-positive cells were stained brown, before Ultra View COPPER was used to treat washed slides for 4 min at 37 °C. After rinsing, the slides were incubated in hematoxylin for 4 min at 37 °C. Finally, the rinsed slides were exposed to BLUING REAGENT for 4 min at 37 °C before they were washed again. AhR-positive cells were observed under a microscope at a magnification of × 200 (Vectra, PerkinElmer, Waltham, MA, USA).

Spleen and liver sections were fixed in 4% formalin and paraffin-embedded (FFPE). Immunohistochemical single staining of EBNA2 (clone PE2, Abcam) and LANA (clone LN53, Clinisciences) was carried out on a Leica BOND-III automated immunohistochemistry system using diaminobenzidin (DAB) as chromogen (Zytomed Systems, Berlin, Germany). In situ-hybridization for the detection of EBER was performed as described previously (Meyer et al., 2011 (link)) with DAP as substrate. Multiplex immunofluorescence (IF) staining was performed on FFPE tissue using Opal dyes and Spectral DAPI (FP1490) from PerkinElmer. Opal dyes 520, 540, 620 and 690 were used to detect EBNA2 (clone PE2, Abcam), CD20 (clone SP32, Cell Marque), LANA (clone LN53, CliniSciences) and IRF4/Mum1 (clone: MUM1p, CliniSciences), respectively. Single stains were used to compensate overlapping spectras. Staining was quantified on a Vectra3 automated quantitative pathology imaging system using Vectra, Phenochart (v1.0) and InForm (v2.4.8) software (all from PerkinElmer), as previously described (Murer et al., 2018 (link)).