Envision flex hrp reagent

IHC was carried out on HCC TMAs according to a previous publication [46] . Briefly, after pre-treating at pH = 6 and blocking with peroxidase, the sections were further incubated with the primary antibody against CA2 (1:50; catalog No. ab226987, Abcam) for 0.5 h. The sections were then treated with EnVision FLEX/HRP reagent (Catalog No. K8000, Dako, Glostrup, Denmark) for 20 min, washed three times, treated with the EnVision FLEX-DAB chromogen (Catalog No. DM827, Dako) for 3 min, and stained by Mayer’s hematoxylin (Lille’s Modification) for 3 min. They were then washed in distilled water for 5 min. All pathological sections underwent double-blind scoring as follows: negative (0), weak (1), strong (2), or very strong (3) by two different pathologists double-blindly. A score of 0 or 1 indicates low expression, and a score of 2 or 3 indicates high expression.

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.