*2.4. Immunohistochemistry*

Formalin-fixed paraffin-embedded (FFPE) sections of bronchial biopsy tissues on slides were obtained from non-asthmatic control individuals, mild asthma, moderate asthma and severe asthma subjects. These slides were archived at the Biobank of the Quebec Respiratory Health Research Network Canada with MUHC REB number BMB-02-039-t [23]. According to retrieved data, patients had been classified into mild, moderate and severe based on frequency of exacerbations, lung function and medication usage, as previously described [23]. Immunohistochemical staining was performed to determine the expression and distribution of Bcl10 as previously described [24]. Briefly, routine deparaffinization in xylene and rehydration steps in decreasing concentrations of ethanol were performed. Heat-activated antigen retrieval was carried out using Tris EDTA buffer at pH 9.0 for Bcl10, as per manufacturer recommendations. The sections were incubated in hydrogen peroxidase blocking solution for 30 min to block the endogenous peroxidase activity. The slides were then blocked in 1% BSA for 20 min at RT and immunostained using mouse monoclonal anti-Bcl10 (Santa Cruz, TX, USA, Cat. No. sc-5273) antibody overnight at 4 ◦C. The slides were developed using HRP/DAB (ABC) Detection IHC kit (Abcam, Cambridge, UK, Cat. No. ab64264), according to manufacturer recommendations. The primary antibody was omitted to serve as

technical negative control and appropriate positive control tissue was used. Nuclei were counterstained blue with hematoxylin (Thermo Scientific Shandon, Waltham, MA, USA). The stained slides were then examined and analyzed by a histopathologist.
