Rabbit anti vegf

Total proteins of rat penis, ADSCs, and 3-day-old MTs were extracted by mechanical homogenization in lysis buffer containing protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Proteins were quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Waltham, MA, USA). A volume of 20 μg sample of each protein was electroseparated in a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Millipore Corporation, Bedford, MA, USA). For incubation, the primary antibodies were rabbit anti-NGF (1:400), rabbit anti-nuclear factor kappa-B (NF-κB, 1:2000), rabbit anti-VEGF (1:200), rabbit anti-α-SMA (1:2000), rabbit anti-nNOS (1:400), mouse anti-tumor necrosis factor-induced protein-6 (TSG-6, 1:200; all from Abcam, Cambridge, UK), and mouse anti-glyceraldehyde phosphate dehydrogenase (GAPDH, 1:10 000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation with the appropriate secondary antibody (1:2000; Santa Cruz Biotechnology), membranes were exposed using chemiluminescent detection reagents. Images were obtained with a C-digit machine (LI-COR Biosciences, Cambridge, UK).

The western blotting analysis was conducted as described previously.^{20 (link)} The primary antibodies used were rabbit anti-VEGF (Abcam, Cambridge, UK), rabbit anti-CD31 (Abcam), rabbit anti-phospho-c-Raf (Ser 338), -phospho-MEK1/2 (Ser 217 and Ser 221), -phospho-ERK1/2 (Thr 202 and Tyr 204), -phospho-PI3K p85 (Tyr458), -phospho-Akt (Thr308), -phospho-mTOR (Ser2481), -phospho-p70S6K (Thr389), -c-Raf, -MEK1/2, -ERK1/2, -PI3K, -AKT, -mTOR, -p70S6K, and -HIF-1α, as well as β-actin (Cell Signaling Technology, Danvers, MA, USA). Primary antibody binding was detected using a peroxidase-conjugated secondary antibody (Pierce, Rockford, IL, USA). The blots were developed by ECL detection regents (GE Healthcare, Waukesha, WI, USA). The gray intensity of protein bands was quantified with ImageJ and normalized to that of β-actin or GAPDH in each sample.

Three days (for interleukin-1β, IL-1β) or seven days (for vascular endothelial growth factor, VEGF) after I/R, five rats from each group were perfused transcardially with saline followed by 4% paraformaldehyde. The brains were removed and post-fixed in 4% paraformaldehyde overnight and then embedded in paraffin. Coronal brain sections (4 μm) were cut for immunohistochemical staining using the standard avidin-biotin-peroxidase complex method. Sections were incubated with rabbit anti-VEGF (1:200; Abcam, Cambridge, UK) or anti-IL-1β (1:200; Santa Cruz Biotechnology, Dallas, Texas, USA) polyclonal antibodies at 4°C overnight, followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (1:200; KPL, Gaithersburg, Maryland, USA) at 4°C for 1 hour. Proteins were visualized using 3,3′-diaminobenzidine and counterstained with hematoxylin. Negative controls were processed in parallel, but the primary antibody was replaced with phosphate-buffered saline. VEGF- and IL-1β-immunoreactive cells were counted in five representative fields of view per section, under an optical microscope at 400× magnification (Olympus, Tokyo, Japan) using Image-Pro Plus 6.0 software (Media Cybernetics Inc., Bethesda, MD, USA).

Proteins were extracted from cardiac lysates as described previously [18 (link)]. Protein samples were subjected to SDS/PAGE, transferred onto a polyvinylidene fluoride membrane. The membranes were incubated in a 5% bovine serum albumin blocking buffer for 1 hour at room temperature and followed by incubation with primary antibodies overnight. The antibodies were used as follows: rabbit anti-total-Akt (Cell signaling; 1: 1000 dilution); rabbit anti-total-eNOS (Affinity; 1: 1500 dilution); rabbit anti-phospho-Akt (Cell signaling; 1: 2000 dilution); rabbit anti-phospho-eNOS (Affinity; 1: 1000 dilution), rabbit anti-VEGF (Abcam 1: 1000; dilution), rabbit anti- cleaved caspase-3 (Cell signaling; 1: 1000 dilution), rabbit anti-α-SMA (Cell signaling; 1: 1000 dilution). Then, membranes were incubated with secondary antibodies conjugated with the horseradish peroxidase for 1 hour at room temperature. Subsequently, the protein signal detection was carried out using chemiluminescence reagent and band intensity were analyzed by Image-Pro Plus 6.