Immunohistochemical Analysis of CD3 and PAX5

Immunohistochemistry for cytoplasmic CD3 and nuclear PAX5 antigens were performed at the Animal Health Laboratory, University of Guelph (an American Association of Veterinary Laboratory Diagnosticians accredited laboratory), using an automated staining instrument (Dako Autostainer, Dako/Agilent, Ontario, Canada). The immunohistochemical reaction for CD3 followed the protocol detailed by Hall et al. (12 (link)). For PAX5, after manual deparaffinization and rehydration, the sections were treated with 3% hydrogen peroxide to quench endogenous peroxidase activity. Heat-induced epitope retrieval was carried out using an EDTA buffer (pH 9) and a pressure cooker device (PT Link, Dako). Primary antibodies against PAX5 (mouse monoclonal, clone 24, Biocare Medical) were diluted 1:50, and incubated at RT for 30 mins. Slides were then incubated with a dual anti-mouse / anti-rabbit anti-IgG horseradish peroxidase–linked polymer (EnVision FLEX, Dako) for 30 mins at RT, and reactions were visualized using Nova Red chromogen (Vector Laboratories, Burlington, ON, Canada). Slides were counterstained with hematoxylin. For CD3 and PAX5 immunohistochemical analysis, thymus, and bursa of Fabricius were used as positive controls, respectively. Negative controls were incubated in the absence of primary antibody. Sections were imaged using the same microscope and camera as described previously.