Alexa fluor 488 conjugated anti mouse igg h l

A mouse monoclonal antibody against M protein and mouse polyclonal antibodies against NP protein and F protein were prepared in our laboratory. Anti-Flag, anti-β-actin, anti-α/β-tubulin, Alexa Fluor 488-conjugated anti-mouse IgG (H+L) and Alexa Fluor 555- conjugated anti-rabbit IgG (H+L) were purchased from Cell Signaling Technology (Beverly, MA, USA). The secondary antibodies against mouse or rabbit used for western blotting were purchased from Bioss Biotechnology (Beijing, China). 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Sigma-Aldrich (St. Louis, MO, USA). CCK-8 cell proliferation and cytotoxicity assay kit were purchased from Solarbio Life Sciences (Beijing, China). CytoD and nocodazole were purchased from Absin Biotechnology (Shanghai, China). Phalloidin-TRITC Conjugate was purchased from AAT Bioquest (Sunnyvale, CA, USA).

The following antibodies were used: mouse IgG1 (MG100) from Life Technologies (Carlsbad, CA), anti-integrin β1 P5D2 (ab24693) from Abcam (Cambridge, UK), anti-tubulin α (T9026) from Sigma-Aldrich, HRP-conjugated polyclonal goat anti-mouse or anti-rabbit antibodies from DAKO Cytomation (Glostrup, DK), and anti-integrin β1 (#4706), anti-MLC2 (#3672), anti-NMHCIIA (#3403), anti-NMHCIIB (#3404), anti-NMHCIIC (#8189), Alexa Fluor^® 488-conjugated anti-mouse IgG (H+L) antibodies from Cell Signaling Technologies (Danvers, MA).

LC3 expressions were analyzed with immunostaining in aortas. The part of aorta with maximum diameter aortas (n = 8 per group) were harvested collected and embedded in Tissu-Tek O.C.T. compound (SAKURA 4583, SAKURA FINETEK USA INC.) The samples were frozen at −80 °C. Then, 5-μm sections were fixed in pre-cold 4% formaldehyde for 15–20 min. After fixation, samples were blocked in 5% BSA in PBS for 1 h. After blocking, samples were incubated with LC3B (E5Q2K) mouse mAb (#83506, Cell Signaling Technology, USA) and rabbit anti-alpha smooth muscle actin antibody (#ab124964, Abcam, Cambridge, USA) at 4 °C for 12 h. The secondary antibodies including Alexa, Fluor 488 conjugated anti-mouse IgG (H + L) (#4408, Cell Signaling Technology, Boston, MA, USA) and Alexa, Fluor 647 conjugated anti-rabbit IgG (H + L) (#4414, Cell Signaling Technology, Boston, MA, USA) were used for detection. Nuclei was stained with 40, 6-diamidino-2-phenyindole, DAPI (Vector Laboratories, Burlingame, CA, USA). ProLong™ Gold Antifade Mountant (Thermo Fisher, P36930) was applied to protect the fluorescence from fading. Images were taken using a Carl Zeiss Axio Imager AX10 with ZEN 2011 (blue edition). The fluorescence was measured using digital image analysis software (ImageJ v.1.41, National Institutes of Health).