Ab96932

Middle cerebral arteries (MCAs) were dissected from 3 animals and pooled in order to isolate primary cerebral vascular smooth muscle cells (CVSMCs) as we have previously described (47 (link), 48 (link)). Human brain microvascular pericytes were purchased (CAP-0030, Angio-proteomie, Boston, MA) and grown in pericyte growth media (CAP-0030). CVSMCs and pericytes were seeded on sterile glass coverslips and positioned inside cell culture plates. Fixation of cells was accomplished with 3.7% paraformaldehyde for 10 min and cells were subsequently treated with 0.1% Triton-100 for 1 h followed by blocking with 0.1% bovine serum albumin for 1 h. Cells were then co-incubated in GPR75 (LSA1589; 1:200) or GPR75 (LSA1589; 1:200) for 24 h at 4°C and then AlexaFluor 555 (A-21428, Fisher; 1:200; 30 min) at RT. Cultured pericytes were co-stained with CD13 (sc-13536, Santa Cruz; 1:200) and CYP4A1 (RPAP-151; 1:200) for 24 h at 4°C followed by AlexaFluor 555 (A32727, Fisher) and Dylight 550 (ab96932, Abcam; 1:200) for 30 min at RT.

A series of parallel sections from animals that were perfused with FITC-dextran was chosen (n = 3 rats), washed in PBS and blocked. The tissue was co-incubated in CYP4A1 (RPAP-151, 1:200) or GPR75 (LSA1589; 1:200) and CD13 (ab108310, Abcam; 1:250) or CD13 (sc-13536, Santa Cruz; 1:200) for 72 h at 4°C followed by secondary incubations in Dylight (SA5-10041, Fisher; 1:200) and Dylight 550 donkey anti-goat (ab96932, Abcam; 1:200) or AlexaFluor 555 (A-21428, Fisher; 1:200) and AlexaFluor 647 (ab150075, Abcam; 1:200) for 30 min. When labeling for NG2, the same protocol was followed, but CD13 and respective secondary incubation steps were omitted and replaced by a primary incubation in NG2 (ab50009, Abcam) followed by the secondary incubation in AlexaFluor 647 donkey anti-mouse at similar concentrations, times and temperatures. Immunostaining of aSMA is described above.