Rabbit anti α smooth muscle actin sma

Western blot analysis was performed as described previously [6 (link)]. In brief, proteins from the CAMs or dissected coronary arteries were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins of these samples were then electrophoretically transferred onto a PVDF membrane (Millipore, USA). The membrane was blocked with 5% nonfat milk and then probed with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-labeled IgG (1:5000). Primary antibodies used were mouse anti-vimentin (1:1000, Abcam), rabbit anti-α-smooth muscle actin (SMA, 1:1000, Abcam), rabbit anti-calponin (1:1000, Abcam), rabbit anti-p62 (1:200, Abcam), mouse anti-ubiquitin (1:1000, Cell Signaling), rabbit anti-CDK1 (1:1000, Cell Signaling) or goat anti-β-actin (1:2000, Santa Cruz). The immuno-reactive bands were detected by chemiluminescence methods and visualized on Kodak Omat X-ray films. Densitometric analysis of the images obtained from X-ray films was performed using the Image J software (NIH).

Tissue preparation and immunohistochemistry were performed as previously described (7 (link)). Cells and tissues were fixed in 4% paraformaldehyde. For cryosection, sections were cut at 12 μm thickness with a Leica CM3050 S cryostat (Leica, Buffalo Grove, IL). For immunohistochemistry, cells and tissues were permeabilized with 0.1% Triton X-100 and blocked with 10% donkey serum for 30 minutes. Primary antibodies included human anti-neuronal nuclear antibody-1 (Hu; 1:16,000; generous gift from Dr. Vanda Lennon), mouse anti-neuronal class III β-tubulin (Tuj1; 1:500; Covance, Dedham, MA), rabbit anti-p75 neurotrophin receptor (P75; 1:500; Promega, Madison, WI), rabbit anti-S100 calcium-binding protein B (S100; 1:100; NeoMarkers, Fremont, CA), and rabbit anti-α-smooth muscle actin (SMA; 1:100; Abcam, Cambridge, MA). Secondary antibodies included donkey anti-mouse Alexa Fluor 488, donkey anti-rabbit Alexa Fluor 546, and donkey anti-human Alexa Fluor 546 (Life Technologies, Carlsbad, CA). Cell nuclei were stained with DAPI (Vector Labs, Burlingame, CA). EdU incorporation was detected using the Click-iT EdU Imaging Kit (Invitrogen, Carlsbad, CA). Images were taken using a Nikon Eclipse TS100 or 80i microscope (Nikon Instruments, Melville, NY).