Muc5ac clone 45m1

Immunohistochemistry was conducted on MucilAir™ inserts fixed with 3.6% formaldehyde/PBS, followed by a dehydration step using 70% ethanol and paraffin inclusion. Tissue sections (4 µm) were incubated with the primary monoclonal antibodies (MUC5AC clone 45M1, FOXJ1 clone 3–19, p63 clone 4A4, Abcam, Cambridge, USA) for 1 hr at room temperature. After 3 washes the biotinylated secondary antibody (Dako, Ely, UK) was used to detect the primary, and visualized using an HRP-conjugated ABC system. Quantitative image analysis was performed using ImagePro Plus6.2 (Media Cybernetics, Cambridge, UK). Twenty images spanning the entire length of the section were acquired per insert to obtain a composite % for MUC5AC, FOXJ1 and p63.

Paraffin-embedded tissues are sliced 4μm thick each section. Deparaffinization is performed by successive incubation in xylene, followed by 100% ethanol and 75% ethanol for 5 min each time. Sections are washed in PBS before being treated with antigen retrieval solution (Citrate buffer pH 6) for 20 min in the microwave (Power 3) and rested for an additional 20 min at room temperature (RT). The sections are then washed in PBS and incubated in blocking solution containing 10% Donkey Serum and Fc block for 1h at RT. Primary antibodies for ClcA1 (clone EPR12254; Abcam, Cambridge, MA) and MUC5AC (clone 45M1; Abcam) are used at 1:100 dilution according to the manufacturer recommendations and incubation is performed overnight at 4°C. Sections are washed in PBS three times before staining with secondary antibodies Donkey anti-mouse and Donkey anti-rabbit (ImmunoJackson Research, West Grove, PA) at 1:500 dilution for 3h at RT. Sections are then washed in PBS three times and nuclear staining is performed using DAPI at 1:10000 dilution for 5 min at RT. Sections are mounted in ProLong Gold Antifade Mountant (Thermo Fisher Scientific, Waltham, MA) and imaged on Nikon Inverted immunofluorescence microscope.