Highly pathogenic avian influenza A virus H5N1 non-structural protein 1 is associated with apoptotic activation of the intrinsic mitochondrial pathway

Bian, Qian; Lu, Jing; Zhang, Li; Chi, Ying; Li, Yan; Guo, Hongxiong

Immunofluorescence staining. NCI-H1299 cells were fixed in 4% (w/v) paraformaldehyde at room temperature for 30 min and permeablized in 0.5% (w/v) Triton X-100, followed by incubation with primary and secondary antibodies for 1 h at room temperature sequentially. Anti-HA serum (AH158; Beyotime Institute of Biotechnology, Haimen, China) with 1:200 was used for the control and Alexa 488-conjugated secondary antibody (1:500, A-11017; Invitrogen; Thermo Fisher Scientific, Inc.) were used to probe for the NS1 protein at room temperature for 1 h. Following protein staining, anti-cytochrome c monoclonal antibody (1:1,000, BD556432; BD Biosciences, Franklin Lakes, NJ, USA) and Alexa 555-conjugated secondary antibody (1:500, A-21427; Invitrogen; Thermo Fisher Scientific, Inc.) were utilized to probe the NCI-H1299 cellular morphology. Finally, 4',6-diamidino-2-phenylindole (DAPI, 1:1,000, D1306; Invitrogen; Thermo Fisher Scientific, Inc.) was used to dye the cell nucleus at room temperature for 1 h. Triple-fluorescence stained cells were observed with a confocal microscope at a high-power magnification of x100 (FV10-ASW, version 01.07.03.00; Olympus Corporation, Tokyo, Japan).