Alexa fluor 488 conjugated anti mouse antibody

FaDu cells were grown on glass coverslips in 6-well plates and treated with 0, 5, or 10 mM metformin for 48 h. The treated cells were fixed with 4% formaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBS at 4 °C for 10 min. The samples were then blocked with 3% bovine serum albumin (BSA) in PBS at 37 °C for 30 min, then stained with anti-LC3B (Cell Signaling Technology no. 3868; diluted 1:200) and Alexa Fluor 488-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA; diluted 1:400). 4′,6-diamidino-2-phenylindole (DAPI) (0.2 μg/mL) was used for nuclear staining. The stained cells were then analyzed under a confocal microscopy (Leica TCS SP8X laser-scanning microscope using a 63 × 1.4 numerical aperture objective) [56 (link)].

293 T cells growing in 24 well plates were transfected with NA plasmids expressing NA of H7N9 SH/2/13 virus. MDCK cells growing in 24 well plates were infected with H7N9 SH/2/13 virus. 293 T and MDCK cells were incubation at 37°C in 5% CO₂ for 48 h. Cells were fixed with 3.7% paraformaldehyde, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, USA), and then incubated with each mAb, followed by incubation with Alexa Fluor^®488 – conjugated anti-mouse antibody (Jackson ImmunoResearch, US). The mAb gH19 (IgG2a) against varicella-zoster virus was used as a negative control. Nuclei were stained with DAPI (Beyotime Biotechnology, China). Cells were then observed under immunofluorescence microscope (Olympus, Japan).

Nonadherent cells were subjected to cytospin, fixed in 4% paraformaldehyde (PFA), and permeabilized with 0.1% Triton X‐100 for subsequent staining as described previously [37 (link)]. Single optical sections were acquired with an SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 63×/1.4 oil immersion objective lens. For the analysis of apoptotic Figures, cells were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and tetramethylrhodamine (TRITC) ‐conjugated Agglutinin (both from Thermo Fisher Scientific). For cytochrome c localization, cells were stained overnight at 4 °C with an antibody against cytochrome c (clone 7H8.2C12, Thermo Fisher Scientific) and counter‐stained with DAPI. Secondary staining was performed with an Alexa Fluor 488‐conjugated anti‐mouse antibody (Jackson ImmunoResearch, West Grove, PA, USA). The total and cytochrome c‐positive cellular areas were calculated from > 20 fields for each sample with an in‐house developed macro for imagej (RRID: SCR_003070). Briefly, the cytochrome c signal from each cell in the field of view was segmented using an automatic threshold (Otsu algorithm) and the area of the signal calculated on the binary images. The cytochrome c area was then normalized by the total cell area, which was identified on the bright‐field transmission image.