Anti smoothelin

Flow cytometry was performed on ADSCs and differentiated cells between 1 and 6 weeks of differentiation. Cells were immunolabeled with hyaluronan receptor (CD44, Millipore, Hessen, Germany), beta-1 integrin (CD29, BioLegend, Fell, Germany), Stro-1 (Invitrogen, Lucerne, Switzerland) and hematopoietic marker (CD34, BD Biosciences, Allschwil, Switzerland) for ADSCs. The differentiation of ADSCs into SMCs was confirmed with the SMC markers anti-calponin (Sigma, Buchs, Switzerland), anti-smoothelin (Santa Cruz, Heidelberg, Germany), anti-MyH11 (Santa Cruz), and anti-alpha-smooth muscle actin (Sigma, Buchs, Switzerland).

Isolated vessels were incubated in tri-sodium citrate buffer 10 mM, pH 6 and heated in a microwave (900W) for 20s for antigen retrieval. Primary antibodies were diluted 1:200 in phosphate buffer (PB: 0.1-M NaH 2 PO 4 and 0.1-M Na 2 HPO 4 ), donkey serum 2%, triton 0.2%, BSA 0.1%, and incubated overnight at 4 C. Anti-PS1 (antibody registry: AB_2170605) and anti-PS2 (antibody registry: AB_325093) were from Thermo Scientific. Anti-MLC2 (myosine light chain 2 specific of smooth muscle, antibody registry: AB_2148039) was from Clinisciences. Anti-smoothelin (antibody registry: AB_2193677) was from Santa Cruz. Anti-NeuN (antibody registry: AB_2149209) was from Millipore. Anti-GFAP (antibody registry: AB_2532994) was from Thermo Fischer. Secondary antibodies (from Clinisciences) were incubated 2 hours at room temperature and washed 3 times. Slices and vessels are mounted in Vectashield hard-set or Fluoromount and observed with a confocal microscope (LEICA SP5) or macroscope (LEICA LSI).