Serum free block solution

hUSCs were plated at 2000 cells/cm² in smooth muscle differentiation media containing equal amounts of high-glucose DMEM and embryonic fibroblast medium (EFM) with 10% FBS, 2.5 ng/mL of transforming growth factor beta1 (TGF-β1), and 5 ng/mL of platelet-derived growth factor BB (PDGF-BB) (R&D Systems, Minneapolis, MN). Cell morphology was recorded before and after growth factor additions for up to 14 days. The slides were fixed with freshly prepared 4% paraformaldehyde for 20 min followed by permeabilization with 0.1% Triton-X100 in PBS for 10 min and blocked with serum-free block solution (Dako, Denmark) for 15 min. Lineage-specific primary antibodies, rabbit polyclonal antibodies to desmin (abcam, Cambridge, MA; catalog number ab15200) and MyoD (C-20, Santa Cruz Biotechnology, Inc., Dallas, TX; catalog number sc-304), were incubated at 4°C overnight followed by secondary antibody conjugated to Alexa Fluor 594 (Life Technologies, Grand Island, NY; catalog number A11072) for 30 min in the dark. The slides were mounted using anti-fade mounting media (Vector Laboratories, Inc., Burlingame, CA) containing propidium iodide (PI) and images were captured using a Leica upright microscope (DM 4000B, Wetzlar, Germany).