Bh3 mjl

Ethical approval was obtained from Renmin Hospital, Hubei University of Medicine. Male C57BL/6 mice (4-5 g, 1 week old, n=20) were purchased from the Animal laboratory of Renmin Hospital. C57BL/6 mice of model group were injected with 2 mg/kg of lipopolysaccharide (LPS, intraperitoneally) for 12 h under anesthesia with 50 mg/kg sodium pentobarbital as reported previously by Fang et al (18 (link)). C57BL/6 mice of sham group were injected with 50 mg/kg sodium pentobarbital. Then, mice were sacrificed via decollation whilst anesthetized. Lung tissue samples were collected and fixed with 4% paraformaldehyde for 24 h at room temperature or stored at -80°C.
Lung tissue samples fixed with paraformaldehyde were paraffin-embedded. The samples were cut into 10-µm sections using a paraffin slicing machine (RM2235; Leica Microsystems GmbH), stained with hematoxylin and eosin for 10 min at room temperature, and observed under a light microscope (magnification, ×100, BH3-MJL; Olympus Corporation).

Cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were blocked with 5% bovine serum albumin (Beyotime Institute of Biotechnology) and 0.1% Triton X-100 for 1 h and incubated with anti-IRAK-1 (Santa Cruz Biotechnology, Inc.) at 4°C overnight. Cells were washed with PBS and incubated with goat anti-rabbit IgG-CFL 555 (Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Cells were washed with PBS and stained with DAPI for 15 min at room temperature. Then, cells were washed with PBS and observed under a light microscope (magnification, ×100, BH3-MJL; Olympus Corporation).