Mouse anti abca1

Frozen tissue samples were sectioned from the aorta at the origins of the aortic valve leaflets (5-µm thickness) in a cryostat. Then each sample was fixed for 20 min in 4% paraformaldehyde solution in PBS followed by permeabilization with 0. 1% Triton X-100 in PBS for 20 min. After washing three times with PBS, sections were incubated with 10% goat serum in PBS for 1 h at room temperature to block nonspecific binding. Then the sections were incubated overnight at 4°C with primary antibody either rabbit anti-SRA1 (1:200) (abcam), mouse anti-ABCA1 (1:200) (abcam), or mouse anti-CD68 (1:200) (abcam) in 2% BSA in PBS. After washing three times with PBS, the sections were incubated for 1 h in 2% BSA in PBS containing a 1:500 dilution of the appropriate secondary antibody, either a goat anti-rabbit IgG conjugated with Chromeo™ 546 or goat anti-mouse IgG conjugated with Alexa Fluor^® 488 (abcam) or goat anti-mouse IgG conjugated with Chromeo™ 546 (abcam). The sections were then incubated with Hoechst for 5 min to stain the cell nuclei after washing three times with PBS. The sections were again washed three times with PBS and mounted with glycerol followed by observing on a ZEISS LSM 710 Laser scanning confocal microscope or common fluorescence microscope. The confocal images were analyzed with ZEN 2009 Light Edition software.

Lysates, or cell fractions were incubated in Laemmli buffer at 55 °C for 10 min under reducing conditions. A total of 30 μg of protein from cell lysates or one-third of the volume collected in cell fraction preparations was separated by SDS-PAGE in 4–20% gradient gels (Biorad) and proteins were then transferred to PVDF membranes. After blocking with 5% TBS-milk, membranes were probed with primary antibodies overnight at 4 °C. The following primary antibodies were used: mouse anti-ABCA1 (Abcam; 1:1000), rabbit anti-GAPDH antibody (Millipore; 1:10,000), mouse anti-actin (CP01, Millipore 1:10,000), anti-MEK, anti-ERp72, anti-V5 and anti-Na⁺/K⁺ ATPase (Cell Signaling; all 1:1000), and rabbit anti-OSBPL7 (Sigma; 1:1000). After washing, membranes were incubated with anti-rabbit or and anti-mouse IgG-HRP antibodies (Promega, 1:10,000). Signals were detected after incubation with Westernbright ECL HRP substrate (Advansta) and luminescence signals were captured with Azure C600 Gel Imaging workstation (Azure Biosystems Inc, USA) or X-ray films.