Anti cftr

The anti-CFTR antibodies used were sc-10747, anti-CFTR (Santa Cruz Biotechnology, Dallas TX), and anti-CFTR MAB3484 (Millipore, Etobicoke, ON Canada). The COX1 antibody used was the anti-COX1 antibody EPR5866 (AB109025 Abcam Cambridge, UK), and the COX-2 antibody used was the anti-COX-2 antibody EPR12012 (AB179800 Abcam Cambridge, UK) and hemagglutinin tag (H-9658 Sigma St. Louis, MO) was used.

Paraffin embedded tissues were sliced and stained with haematoxylin and eosin. For each animal, ten randomly chosen microscopic fields were investigated under a light microscope. For quantification of oedema, necrosis and leucocyte infiltration, a modified histopathologic score adapted from Kyogoku et al ³⁹ was used, that scored 0 for absent, 1 for less than 20%, 2 for 20%‐50% and 3 for more than 50% as previously described.⁴⁰ Immunohistochemistry was performed from paraffin embedded tissue samples as previously reported. The following primary antibodies were used in a dilution of 1 to 200: anti‐CFTR (Clone: A‐3, Santa Cruz Biotechnology, sc‐376683), p67‐phox (Santa Cruz Biotechnology, sc‐374510) and anti‐CD11b (abcam, ab133357). For immunofluorescence staining of CFTR snap‐frozen tissue embedded with Tissue‐Tek® OCT^TM compound (Sakura Finetek, Alphen aan den Rijn) was cut into 1‐ to 2‐µm‐thick slices, fixed with 20% acetone and washed with PBS. The anti‐CFTR antibody (Clone: M3A7, DLN‐06996, Dianova) was used in a 1:100 dilution in 20% FCS, and incubation was performed over night at 4°C. An anti‐rat IgG FITC‐labelled antibody (Jackson ImmunoResearch) served as a secondary antibody. Nuclei were stained by DAPI and slides were mounted with DACO mounting medium (Agilent Technologies Inc) for immunofluorescence.

Prostaglandin E₂ (PGE₂) and forskolin were purchased from Sigma. pH-sensitive fluorescent dye, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein acetoxymethyl ester (BCECF-AM), was from Invitrogen. Anti-CFTR, anti-SLC26A6, anti-P38, anti-phospho-P38, and anti-β-actin antibodies were from Santa Cruz. All other chemicals in solutions were obtained from Sigma and Calbiochem.