Renoir red

Double-immuno-labelling experiments were performed on 3-µmthick FFPE sections, using anti-human PAX5 clone DAK-Pax5 (Agilent/Dako, cat. no. M7307, Glostrup, Denmark) and CD20 clone L26 (Agilent/Dako, cat. no. M0755). All incubation steps were performed automatically on the Omnis (Agilent/Dako). Briefly, FFPE sections were exposed to deparaffinization, followed by antigen retrieval using EnVision™ FLEX Target Retrieval solution (3-in-1) pH 9 (Agilent/Dako, cat. no. GV800/821) at 97°C (24 min). In the first sequence, slides were incubated with PAX5 diluted 1:30 in Renoir Red (Biocare Medical, cat. no. PD904, Pacheco, CA, USA) for 30 min (32°C), and reactions were visualized with EnVision™ FLEX /HRP/DAB Detection Reagent (Dako, cat. no. GV800/GV821). After visualization with DAB, slides were incubated with CD20, diluted 1:500 in Envision Flex Antibody Diluent (Agilent/Dako, cat. no. K8006) for 30 min (32°C), reactions were detected with EnVision™ FLEX/HRP Reagent (Dako, cat. no. GV800/GV821) and visualized using Magenta Substrate chromogen system (Agilent/Dako, cat. no. GV925). All slides were counterstained with haematoxylin and mounted with Pertex.

Double-immunofluorescence (dIF) studies (simultaneous technique) were performed on representative cases (5 ES-MF and 2 AS-MF) to investigate whether the neoplastic T-cells and the subpopulation PAX5^–/CD20⁺ cells were malignant T-cells displaying an aberrant CD20 expression. The dIF staining was performed as described in detail elsewhere (26 (link)). In brief, after deparaffinization and antigen retrieval, as described above, the slides were incubated for 30 min (32°C) with a mixture of anti-human CD3 clone EP41 (Cell Marque, cat. no. AC-0004, Rocklin, CA, USA) and anti-human CD20 clone L26 (Agilent/Dako, cat. no. M0755) diluted in Renoir Red (Biocare, cat. no. PD904) 1:20 and 1:250, respectively. Slides were incubated with a mix of goat anti-rabbit conjugated with Alexa Fluor 594 (Fisher Scientific, cat. no. 11012, Roskilde, Denmark) and goat anti-mouse conjugated with Alexa Fluor 488 (Fisher Scientific, cat. no. 11001), both diluted 1:200 in Envision Flex Antibody Diluent (Agilent/Dako, cat. no. K8006), for 30 min (32°C). After washing, the slides were air-dried and mounted with Vectashield with DAPI (Vector Labs, cat. no. H1200, Burlingame, CA, USA). Slides were evaluated with a Nikon Eclipse 80 fluorescence microscope with standard sets of filters for each fluorochrome.