Fig 2: Classic Hodgkin lymphoma stained using a combination of tyramide-barcodes and barcode-conjugated primary antibodies.a CD30 (1:50 dilution, mouse host, clone Ber-H2, Invitrogen) and MUM1 (1:100 dilution, mouse host, clone MUM1p, Agilent Dako Products) staining was performed using the tyramide-barcodes, which stains the cell membranes and nuclei of Hodgkin cells, respectively (scattered T cells also express MUM1). Other antigens were stained using commercially available barcode-conjugated primary antibodies (Akoya Biosciences, 1:200 dilutions except for CD3e, which was 1:100 dilution). DAPI = blue, CD30 = magenta, MUM1 = yellow, CD68 = red, CD3e = cyan. Staining for CD4, CD8, CD11c, CD20, CD45RO, CD107a, and Ki67 is shown in Fig. 4. b Virtual H&E staining was generated using DAPI and eosin fluorescence images collected after the regular CODEX imaging24. Circles help to identify Hodgkin/Reed-Sternberg cells seen in the fluorescence image and corresponding virtual H&E image.

Fig 3: Additional images of classic Hodgkin lymphoma demonstrating the various stains performed.a CD30 = magenta, MUM1 = yellow, DAPI = blue, CD3e = cyan, CD68 = red. b CD30 = white, CD3 = yellow, CD20 = blue. c CD30 = white, CD8 = yellow, CD4 = red, CD20 = blue. d CD30 = white, CD107a = yellow, CD45RO = magenta. e CD30 = white, CD68 = magenta, CD107a = yellow, CD8 = cyan. CD107a is seen variably expressed in CD68+ histiocytes. CD107a puncta are also seen in association with CD8+ cells, though some other puncta are also seen which are of uncertain significance and might indicate non-specific staining. f CD30 = white, CD3e = yellow, CD20 = blue, Ki67 = red. g CD30 = white, CD68 = magenta, CD11c = yellow. CD11c expression is distributed in a patchy manner in the imaged tissue and primarily found in region 2 (compare with Supplementary Fig. 10; a CD11c rich area is shown here) and associated more frequently with helper T cells and histiocytes. Images were created using the Akoya CODEX MAV plugin for ImageJ.