Ng2 chondroitin sulfate proteoglycan

After washing several times with PBS, samples were then immersed in 3% bovine serum albumin (BSA) for 1 hour at room temperature to minimize nonspecific binding of antibodies. Samples were incubated with antibodies to CD31 (an endothelial cell marker; Millipore, Temecula, CA, USA; 1:50; Cat #MAB1398Z), fluorescein isothiocyanate-conjugated antibody to smooth muscle α-actin (a smooth muscle cell marker; Sigma-Aldrich, St. Louis, MO, USA; 1:200; Cat #F3777), smooth muscle α-actin (a smooth muscle cell marker; Abcam, Cambridge, UK; 1:200; Cat #ab5694), NG2 chondroitin sulfate proteoglycan (a pericyte cell marker; Millipore; 1:50; Cat #ab5320), and βIII-tubulin (a neuronal marker; Abcam; 1:100; Cat #ab107216) for 48 hours at 4℃. After washing several times with PBS, the samples were incubated with Donkey Anti-Mouse IgG H&L Alexa Fluor® 405 (Abcam; 1:200; Cat #ab175658), rhodamine AffiniPure Goat Anti-Armenian Hamster IgG (H+L) (Jackson ImmunoResearch, West Grove, PA, USA; 1:50; Cat #127-025-160), rhodamine Affinipure Donkey Anti-Chicken IgY (H+L) (JacksonImmuno Research; 1:50; Cat #703-025-155). Primary and secondary antibodies were diluted in PBS containing 0.1% triton X-100 and 3% BSA (Sigma-Aldrich). Samples were mounted in a solution containing 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc., Burlingame, CA, USA) for nuclei staining.

To determine cell type, cells were stained with antibody to von Willebrand factor (vWF, an EC marker; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:50), CD34 (an EC marker; Abcam, Cambridge, MA, USA; 1:50), NG2 chondroitin sulfate proteoglycan (a pericytes marker; Millipore, San Francisco, CA, USA; 1:50), platelet-derived growth factor receptor-β (PDGFR-β, a pericytes marker; Santa Cruz Biotechnology; 1:50), fibroblast-specific protein 1 (FSP1, a fibroblast marker; Santa Cruz Biotechnology; 1:50), or DAPI (a nucleus marker; Vector Laboratories Inc., Burlingame, CA, USA). Signals were visualized and digital images were obtained with a confocal microscope (FV1000; Olympus, Tokyo, Japan).

To characterize cell types, the cells were cultured on sterile cover glasses (Marienfeld Laboratory, Lauda-Königshofen, Germany), which were placed on the bottom of 12-well plates and grown until nearly confluent. Then, the cells were stained with antibodies to CD31 (an EC marker; Chemicon, Temecula, CA, USA; 1:50), CD90 (a fibroblast marker; R&D Systems Inc.; 1:50), NG2 chondroitin sulfate proteoglycan (NG2; a pericyte marker, Millipore, San Francisco, CA, USA; 1:50), smooth muscle α-actin (α-SMA, a smooth muscle cell marker; Sigma-Aldrich; 1:100), or DAPI (a nucleus marker; Vector Laboratories Inc., Burlingame, CA, USA) as previously described [10 (link)]. Signals were visualized, and digital images were obtained with a confocal fluorescence microscope (K1-Fluo; Nanoscope Systems, Inc., Daejeon, Korea).