Ab232761

The arteries of ApoE-/-and C57BL/6 mice were collected for histological and immunochemical analysis. All the samples were embedded in optimum cutting temperature compound after fixing with 4% paraformaldehyde overnight. A series of cryosections (6-μm thick) were cut, and the expression of FSTL3 was probed with a specific antibody (1:50, Abcam: #ab232761, Cambridge, United Kingdom). Subsequently, the sections were incubated in a horseradish peroxidase (HRP)-conjugated secondary antibody (1:100) for 1 hour, and then chromogenic detection was performed using 3, 3′-diaminobenzidine. Finally, the sections were counterstained with hematoxylin.
For immunofluorescence analysis, FSTL3 (1:50, Abcam: #ab232761) and CD68 (1:50, Abcam: #ab31630) antibodies were used. After incubation with anti-FSTL3 and CD68 for 12 hours at 4°C, the sections were immunostained with secondary antibodies conjugated with Alex 549 and 488 (1:1000). All the sections were visualized under a microscope (Olympus Microsystems) and were photographed.

Total proteins were extracted by lysing the cells in the ProteoJET Mammalian Cell Lysis Reagent (Fermentas, MD), and 20 µg was resolved by electrophoresing on a 12% SDS-polyacrylamide gel. The proteins were then blotted on a poly (vinylidenedifluoride) membrane and probed with antibodies against FSTL3 (1:500, Abcam: #ab232761), CD36 (1:1000, Abcam: #ab133625), LOX-1 (1:1000 Abcam: # ab60178), and β-actin (1:2000, Abcam: #ab8226) by overnight incubation at 4°C. Thereafter, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000) for 1 hour at 25°C. Finally, the blot was developed using an ECL detection system (Millipore, Burlington, MA), and the intensity of the bands was quantified by densitometry using Quantity One software (Bio-Rad, Hercules, CA).