Texas red anti mouse igg

C17.2 cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature and then permeabilized by incubating with Triton X-100 (0.1% v/v in PBS) for 10 minutes and blocked in 1% BSA/PBS for 30 minutes. Thereafter, BSA/PBS solution was aspirated and replaced with primary antibodies, diluted in 1% BSA/PBS. The primary antibodies used were: Mouse anti-nestin (Rat-401, 1 μg/ml, developed by S. Hockfield, obtained from DSHB, developed under auspices of NICHD, maintained by University of Iowa) (1:50); Rabbit anti-GFAP (Dako) (1:100); and Rabbit anti-α-βIII-tubulin (Cell Signalling Technology) (1:100). Cell samples were incubated with these primary antibodies for 2.5 hours. The primary antibody solution was then removed and cells washed with PBS (5 times). Secondary antibodies, Texas Red, anti-mouse IgG (Vector laboratories) (1:200); Texas Red, anti-rabbit IgG (Vector laboratories) (1:200); and Alexa flour 486 anti-rabbit IgG (Life Technologies) (1:400), were diluted according to manufacturer’s instructions in 1% BSA/PBS and then applied to the cells for 1 hour. The secondary antibody solution was then aspirated and cells washed with PBS extensively. Finally, cells were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI).

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).