Anti human αsma

Proteins were extracted using NucleoSpin RNA/protein (Macherey-Nagel) and quantified by the Bicinchoninic acid method. For each experimental condition, 30 μg of proteins were separated by electrophoresis on Bis-Tris gel (GenScript Biothec, Leiden, Netherlands) and transferred onto Hybond-C-nitrocellulose membranes (Life Technologies Ltd. Paisley, UK).
After 1 h in blocking solution (SuperBlock Blocking buffer in PBS, Thermo Scientific), membranes were incubated overnight at 4 °C with the following primary antibodies: anti-human αSMA (dilution 1:300, Sigma-Aldrich), S100A4 (dilution 1:100, Santa Cruz Biotechnology, Dallas, USA), COL1 (dilution 1:600, Proteintech), and FN (dilution 1:2,000, Sigma-Aldrich). The membranes were subsequently incubated with (HRP)-conjugated secondary antibodies (dilution 1:2,000; Cell Signaling, MA, USA). To confirm similar loading of protein samples into the gels and the efficiency in the electrophoretic transfer, membranes were incubated with primary HRP-conjugated antibody to human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (dilution 1:2,000; Santa Cruz Biotechnology). Protein synthesis was detected using the enhanced chemiluminescence system (Luminata Crescendo, Millipore), and the densitometric analysis was performed by the UVITEC Image Analysis System (UVITEC, Cambridge, UK).

Culture chamber slides were coated with fibronectin and seeded with ECFC and bmMPC at a 1:1 ratio. After in vitro co-culture for 7 days, bmMPC differentiate into VSMC/pericytes [43 (link)]. Once differentiated, cells were fixed with cold pure methanol on ice for 10 min. For immunostaining of ECFC, samples were incubated with a mAb anti-human von Willebrand factor (Dako) for 1 h at room temperature, followed by incubation with the secondary antibody Texas Red anti-mouse IgG (Vector) for 1 h at room temperature. Differentiated bmMPCs were incubated with anti-human calponin (Abcam), anti-human Sm22α (Abcam), anti-PDGFRb (Santa Cruz), anti-human NG2 (R&D Systems), anti-human αSMA (Sigma) or a negative control antibody (Santa Cruz). After washing samples were incubated with the appropriate FITC-labeled secondary antibody, and mounted using Vectashield with DAPI (Vector).