Zyla 4.6 scmos camera

Cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, washed with PBS and then incubated with primary antibody for E-cadherin (Cat# af0131, 1:200 dilution; Affinity, Cincinnati, OH, USA) and α-smooth muscle actin (α- SMA) (Cat# 19245, 1:100 dilution; Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. The cells were washed with PBS and incubated with secondary antibody cy3 (AS007, 1:200 dilution; ABclonal) for 1 h at room temperature. For negative control, the cells were incubated with PBS instead of primary antibody. The nuclei were stained with 1 μg/mL DAPI in the dark for 1 min at room temperature, and the slices were sealed with blocking solution containing anti-fluorescence quencher, and observed under fluorescence microscope (CKX53, Olympus) equipped with a Zyla 4.6 sCMOS camera to record the images. The immunolabeling was assessed by ImageJ software analysis program.

Human bronchial epithelial cell line BEAS-2B was purchased from iCell Bioscience, Inc. (Shanghai, China) and stained for Pan Cytokeratin (PCK) for cell identification. Briefly, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 10 min at room temperature. The cells were washed with PBS and then incubated with primary antibody for PCK (Cat# bs- 5352r, 1:100 dilution; Bioss, Woburn, MA, USA) overnight at 4°C, washed with PBS and incubated with secondary antibody cy3 (Cat# AS007, 1:200 dilution; ABclonal, Woburn, MA, USA) for 1 h at room temperature. For negative control, the cells were incubated with PBS instead of anti-PCK antibody. The nuclei were stained with 1 μg/mL 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MA, USA) in the dark for 1 min at room temperature, and the slices were sealed with blocking solution containing anti-fluorescence quencher, and observed under fluorescence microscope (CKX53; Olympus, Tokyo, Japan) equipped with a Zyla 4.6 sCMOS camera to record the images. Immunolabeling was assessed by ImageJ software analysis program (NIH Image, Bethesda, MD, USA).