Muc5ac antibody

SP600125 was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Antibodies against phospho-ERK 1/2 (Thr-202/Tyr204), phospho-p38 MAPK (Thr-180/Tyr-182), phospho-JNK 1/2 (Thr183/Tyr185) and phospho-EGFR (Tyr1068) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Monoclonal antibody against β-actin was purchased from Sigma-Aldrich Japan (Tokyo, Japan). Antibody against γ-tubulin was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). MUC5AC antibody was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Antibody against HO-1 was purchased from Abcam (UK).

PHNECs were plated on 6-well cell culture plates and allowed to adhere overnight, and then treated with and without recombinant human IL-19 (rhIL-19) (300 ng/ml, 1035-IL-025, R&D System, Minneapolis, MN,USA) for 24 h. All plates were washed three times with PBS and fixed for 10 min in 4% paraformaldehyde. Cells were washed three times with PBS and blocked for 30 min in 5% normal goat serum, and then incubated by MUC5AC antibody (mouse monoclonal, 1:50, MA5-12178, Thermo Fisher Scientific) at 4°C overnight. After washing with PBS, cells were incubated with Cy3-labeled goat anti-mouse IgG antibody (1:400, BS10006, Bioworld, St. Louis Park, MN, USA) for 1 h and DAPI (300 nm) for 5 min at room temperature. Immunofluorescence staining was imaged using confocal microscope at ×630 magnification.

All tissues were fixed with 4% paraformaldehyde, embedded in paraffin, cut into 4 μm thick sections and then stained for MUC5AC following routine staining procedures. Sections were incubated overnight at 4°C with MUC5AC antibody (mouse monoclonal, 1:200, MA5-12178, Thermo Fisher Scientific, Waltham, MA, USA). Results were expressed as the intensity of staining and the percentage of IHC-positive cells to total epithelial area. Sections were scored 0, 1, 2, 3 points for colorless, light yellow, brown yellow and brown, respectively. Sections were them scored 0, 1, 2, 3, 4 points for 0, 1–25%, 26–50%, 51–75%, 76–100% of IHC-positive cells to total epithelial area, respectively. The final score was defined as the sum of the previous two scores. Each section had five randomly selected regions evaluated, after which the scores of the five regions were then averaged.