Rabbit anti pdgfrα

The following primary antibodies were used for IHC or western blot analyses: mouse anti-GPR56 (H11) (1:200)40 (link) and rabbit anti-GPR56 (199) (1:200)19 (link), rabbit anti-MBP (Millipore; Cat #AB980, 1:200), rat anti-MBP (Abcam, Cat# ab7349), mouse anti-O4 (Millipore; Cat #MAB345, 1:400), rabbit anti-NG2 (Millipore; Cat #AB5320, 1:200), goat anti-Sox2 (Santa Cruz; Cat #sc-17320, 1:400), rabbit anti-Olig2 (kind gift from Charles Stiles, 1:10,000), rat anti-PDGFRα (BD Bioscience; Cat #558774, 1:500), rabbit anti-PDGFRα (Cell Signaling Technologies; Cat #3164S, 1:500) and rat anti-Ki67 (Affymetrix eBioscience; Cat #14-5698-80, 1:100), rat anti-BrdU (Accurate Chemical and Scientific Corporation; Cat #OBT0030S, 1:100), rabbit anit-PLP (Abcam, Cat #ab28486, 1:1,000), mouse anti-RhoA (Cytoskeleton, Cat# ARH03-A, 1:500), mouse anti-CDK2 (Santa Cruz; Cat #sc-6248, 1:1,000), mouse anti-β-actin (Sigma, Cat #A5044, 1:5,000) and mouse anti-Ki67 (BD Bioscience; Cat #550609, 1:100). Secondary antibodies were goat anti-mouse or anti-rat conjugated with either Alexa 488 (Life Technologies, 1:1,000) or Alexa 546 (Life Technologies, 1:1,000) and goat anti-rabbit conjugated with Alexa 546 or 555 (Life Technologies, 1:1,000), goat anti mouse or rabbit IgG-HRP (Sigma, Cat# A4416 or A6154, 1:3,000).

Analysis of hESC-OPCs was performed by a FACS
Calibur flow cytometer (Becton Dickinson, USA)
with a 488 nm argon laser. Briefly, the cells were
dissociated with accutase (Millipore, USA) at 37˚C
for 5 minutes. Then, cells were washed twice with
PBS by centrifugation at 1500 rpm for 10 minutes.
The cells were fixed with 4% paraformaldehyde
in PBS for 10 minutes. After washing twice with
PBS, the cells were permeabilized with 0.1% triton
X-100 for 15 minutes. Then, cells were washed twice
and triturated with a narrow glass Pasteur pipette
to prepare a single cell suspension. The primary
antibody, rabbit anti-PDGFRα (1:200, Cell Signaling,
USA), was added to the cells and the suspension was
allowed to incubate at 37˚C for 2 hours. A secondary
antibody, goat anti-rabbit IgG-FITC (1:50, Chemicon,
USA), was added to the cells, after which they were
incubated at 37˚C for 45 minutes. The negative
control was the sample without primary antibodies.
Analysis of annexin V/PI staining by flow cytometry
was performed as previously described. A forward
and side scatter gate was used to select target cells
from the aggregates. We calculated a total of 10000
events for each sample with data analysis by WinMDI
2.9 software. Green fluorescence was detected by the
FL1-H detector and displayed in the histogram.

Cultured cells or brain slices (30 μm) were fixed with 4% paraformaldehyde followed by three washes in PBS and permeabilized with 0.1% Triton X-100 for 10 min (except for O4, O1 immunostainning) then blocked in 10% goat serum (Invitrogen Corp. USA) for 1 h following incubation with the primary antibody overnight at 4 °C. Primary antibodies were diluted in blocking solutions as follows: mouse anti-MBP (Biolegend, USA; 1:500), mouse anti-APC, CC1 clone (Millipore, USA; 1:500), mouse anti-O4 (R&D Systems Inc, USA, 1:800), mouse anti-O1 (R&D Systems Inc, USA, 1:800), rabbit anti-PDGFRα (Cell Signalling Technology, USA; 1:500), rabbit anti-glial fibrillary acidic protein (GFAP, Biolegend, USA; 1:500) mouse anti-BrdU (Sigma St Louis, USA; 1:500). The tissue or cells were then washed with PBS and incubate in Alexa Flour-conjugated secondary antibodies (Invitrogen Corp. USA; 1:500) for 1 h. Images were obtained using Olympus photomicroscope and analyzed using Image-Pro plus 6.0 software.

SMCs were lysed in RIPA buffer containing protease inhibitors (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, and 10 μg/ml aprotinin). Protein concentrations of cell lysates were determined using a Bio-Rad DC™ Protein Assay kit. Approximately 15–30 μg of proteins from each sample were separated on 4–20% Mini-PROTEAN TGX precast gels (Bio-Rad) and transferred to a PVDF membrane. Proteins of interest were detected by immunoblotting using the following primary antibodies and dilution ratios: Rabbit anti-BRD4 (1:1000) from Abcam (ab128874), rabbit anti-PDGFRα (1:1000) from Cell Signaling technology (3164 s), rabbit anti-PDGFRβ (1:200) from Santa Cruz (sc-432) and mouse anti-β-actin from Sigma-Aldrich. After incubation of the blots with HRP-conjugated secondary antibodies (1:3000 for goat anti-rabbit or 1:10,000 for goat anti-mouse, Bio-Rad), specific protein bands on the blots were visualized by applying enhanced chemiluminescence reagents (Pierce) and then recorded with a LAS-4000 Mini imager (GE, Piscataway NJ). Band intensity was quantified using ImageJ.

Whole-liver lysates were prepared in Triton-X 100 lysis buffer containing protease inhibitors and protein concentrations were quantified using the Bradford method with bovine serum albumin (BSA) as the standard. Resolution by SDS-PAGE was followed by immunoblotting using the following antibodies: rabbit anti-PDGFRα (#3164), rabbit anti-phospho-p44/42 MAPK (#9101), rabbit anti-p44/42 MAPK (#9102), rabbit anti-β actin (#4(a967), all from Cell Signaling (Danvers, MA) and anti-phospho-Smad3 (#ab52903) from Abcam (Cambridge, MA). Epitope-primary antibody complexes were detected using species-specific secondary antibodies conjugated to horseradish peroxidase (HRP) followed by enhanced chemiluminescence (ECL) (Thermo Fisher Scientific Pierce, IL).

The antibodies used were as follows: rat anti-Ki67 (#14-5698-82, Thermo Fisher Scientific, 1:500), rabbit anti-Olig2 (#AB9610, Millipore, 1:400), rat anti-MBP (#MCA409S, Bio-Rad Laboratories, 1:500), mouse anti-CC1 (#ab16794, Abcam, 1:200), goat anti-PDGFRα (#AF1062, R&D Systems, 1:100), rabbit anti-PDGFRα (#3164 S, Cell Signaling Technology, 1:1000), mouse anti-P65 (#6956 S, Cell Signaling Technology, 1:1000), mouse anti-p-P65 (#3036 S, Cell Signaling Technology, 1:1000), mouse anti-Actin (#M1210-2, HUABIO, 1:5000), mouse anti-Tubulin (#M1305-2, HUABIO, 1:5000), mouse anti-RIPK1 (#610458, BD, 1:1000), rabbit anti-p-RIPK1 (#65746, Cell Signaling Technology, 1:1000). Secondary antibodies: Cy^TM3 affinipure donkey anti-rat IgG (H + L) (#712-165-153, 1:400), Cy^TM3 affinipure donkey anti-mouse IgG (H + L) (#715-165-151, 1:400), Alexa Fluor 488 affinipure donkey anti-rabbit IgG (H + L) (#711-545-152, 1:400) and Alexa Fluor 488 affinipure donkey anti-goat IgG (H + L) (#705-545-003, 1:400) were purchased from Jackson ImmunoResearch; Goat anti-mouse IgG (H + L) conjugated with HRP (#31430, 1:20000), goat anti-rabbit IgG (H + L) conjugated with HRP (#31,460, 1:20000) were purchased from Thermo Fisher Scientific.