Inverted immunofluorescence microscope

5-Ethynyl-20-deoxyuridine (EdU) incorporation assay was performed using by Clict-ITTM EdU (Thermo, C10420) according to the manufacturer’s instructions. The cells were fixed in 4% paraformaldehyde (Sigma, USA), briefly incubated with 20 uM EdU for 1 h, and incubated in PBS + 3%BSA. Then, click-iT reaction reagents were used to incubate the cells for 0.5 h. Blocking with 3% BSA/0.05% Tween-20/PBS (PBST block buffer) was carried out for 1 h at room temperature. The cells were incubated with a primary antibody—anti-Biotin (Rabbit, Abcam, ab53494)—diluted in blocking solution for 2 h overnight at 4°C, washed three times with PBST, incubated with secondary antibody—Anti-Rabbit IgG Alexa Fluor 647 (Invitrogen, A31573)—in the same solution for 1 h at RT, and washed three times with PBST. To the second wash, 0.1 μg/ml DAPI was added. The cells were observed using an inverted immunofluorescence microscope (Nikon) at ×40 magnification.

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.