Confocal laser scanning microscopy

After removal of the medium, neurons on coverslips were washed twice with PBS, and then incubated with 5 μL annexin-V-FITC and 5 μL propidium iodide (KeyGEN Biotech, Nanjing, China) in 500 μL binding buffer in the dark at room temperature for 5 minutes. After mounting, neurons were observed by confocal laser scanning microscopy (Bio-Rad). In the early stage of apoptosis, the cell membrane displayed green fluorescence, and the nuclei were not stained. In the middle and late stages of apoptosis, the cell membrane displayed green fluorescence, and the nuclei were stained red. Nuclei of dead disintegrated cells were stained red. Fluorescence images were captured using LaserSharp 2000 software (4.5.3; Bio-Rad). A total of 20 images were randomly obtained from each group for measurement of fluorescence intensity (Niu et al., 2011).

After removal of the medium, neurons were washed twice with 0.01 M PBS (pH 7.4) at 37°C, fixed with 4% paraformaldehyde for 30 minutes, washed twice with PBS, dried, and then blocked with normal serum (Life Technologies) (diluted with 0.01 M PBS at 1:10) at room temperature for 20 minutes. Excess liquid was shaken off. Neurons were incubated with rabbit anti-Bcl-2 or Bax polyclonal antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. PBS was used as negative control. After washing with PBS, neurons were incubated with goat anti-rabbit IgG-FITC (1:100; Boster, Wuhan, China) at 37°C for 30 minutes. Neurons were then washed, mounted with glycerol in PBS (1:1), and finally observed by confocal laser scanning microscopy (Bio-Rad). Scanning parameters were as follows: excitation wavelength, 554 nm; observation wavelength, 575 nm; 40× objective; point scanning; zoom (1.0). Scanned images were photographed and analyzed using LaserSharp 2000 software (v. 4.5.3; Bio-Rad) (Zhang et al., 2007a, 2008).

Lungs were fixed by immersion in 10% neutral-buffered formalin and embedded in paraffin. The slides with lung sections (5 µm) were soaked in xylene and rehydrated in 100, 95, 85 and 70% solutions of ethanol for 5 min. Following blocking with 10% fetal calf serum for 1 h at room temperature, the sections were incubated with rabbit polyclonal primary antibodies against α-, β- or γ-ENaC (1:200; sc-21012, sc-21013 and sc-21014 respectively; Santa Cruz Biotechnology, Inc.) overnight at 4°C. The tissues were then incubated with a goat anti-rabbit secondary antibody labeled with Alexa Fluor 594 (1:400; A-11012; Thermo Fisher Scientific, Inc.) at 37°C for 1 h. Subsequently, nuclei were stained with DAPI (1:2,000; Sigma-Aldrich; Merck KGaA). Images were captured by confocal laser scanning microscopy (Bio-Rad Laboratories, Inc.) and analyzed using Image-Pro Plus software, version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).