Ab204131

Immunofluorescence staining was performed with 4-μm paraffin cross-sections from mouse abdominal aorta as well as with paraformaldehyde-fixed cells. After deparaffinization with xylene and rehydration, the slides were blocked by pre-incubation with 10% normal goat serum (KPL, 710027) for 1 h. Paraformaldehyde-fixed VSMCs were permeabilized by incubation with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 30 min. Then, the tissue section or cells were incubated overnight at 4°C with the following primary antibodies: anti-α-SMA (1:50, Santa Cruz Biotechnology, sc-130617), anti-Beclin1 (1:100, Abcam, ab207612), anti-LC3B (1:100, Abcam, ab48394), anti-KLF3 (1:50, Santa Cruz Biotechnology, sc-514500), anti-NEDD4L (1:100, Abcam, ab46521), and anti-PFKP (1:100, Abcam, ab204131). Secondary antibodies were rhodamine-labeled antibody to rabbit immunoglobulin G (IgG) (1:50, KPL, 031506) and fluorescein-labeled antibody to mouse IgG (1:50, KPL, 021815), or rhodamine-labeled antibody to mouse IgG (1:50, KPL, 031806) and fluorescein-labeled antibody to rabbit IgG (1:50, KPL, 021516), and 4′,6-diamidino-2-phenylindole (DAPI) (1:50, MP Biomedicals, 157574) was used to stain nuclei in each experiment. Images were captured by confocal microscopy (DM6000 CFS, Leica Microsystems) and processed by LAS AF software.